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(The FASEB Journal. 2006;20:1209-1211.)
© 2006 FASEB

Granzyme B mediates neurotoxicity through a G-protein-coupled receptor

Tongguang Wang*, Rameeza Allie*, Katherine Conant*, Norman Haughey*, Jadwiga Turchan-Chelowo{dagger}, Katrin Hahn*, Antony Rosen{ddagger}, Joseph Steiner*, Sanjay Keswani*, Melina Jones*, Peter A. Calabresi* and Avindra Nath*,1

* Department of Neurology, Johns Hopkins University, Baltimore, Maryland, USA;

{dagger} Department of Internal Medicine, Johns Hopkins University, Baltimore, Maryland, USA; and

{ddagger} Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky, USA

1Correspondence: Department of Neurology, Pathology Bldg. Rm. 509, 600 N. Wolfe St., Baltimore, MD 21287, USA. E-Mail: anath1{at}jhmi.edu

ABSTRACT

Neuroinflammatory diseases such as multiple sclerosis (MS) are characterized by focal regions of demyelination and axonal loss associated with infiltrating T cells. However, the role of activated T cells in causing neuronal injury remains unclear. CD4 and CD8 T cells were isolated from normal donors and polyclonally activated using plate-bound anti-CD3 and soluble anti-CD28. The conditioned T cell supernatants caused toxicity to cultured human fetal neurons, which could be blocked by immunodepleting the supernatants of granzyme B (GrB). Recombinant GrB also caused toxicity in neurons by caspase-dependent pathways but no toxicity was seen in astrocytes. The neurotoxicity was independent of perforin and could not be blocked by mannose-6-phosphate. However, GrB-induced neurotoxicity was sensitive to pertussis toxin, implicating the stimulation of Gi{alpha} protein-coupled receptors. GrB caused a decrease in cAMP levels but only modest increases in intracellular calcium. The effect on intracellular calcium could be markedly potentiated by stromal-derived factor 1{alpha}. GrB-induced neurotoxicity could also be blocked by vitamin E and a neuroimmunophilin ligand. In conclusion, GrB may be an important mediator of neuronal injury in T cell-mediated neuroinflammatory disorders.—Wang, T., Allie, R., Conant, K., Haughey, N., Turchan-Chelowo, J., Hahn, K., Rosen, A., Steiner, J., Keswani, S., Jones, M., Calabresi, P. A., and Nath, A. Granzyme B mediates neurotoxicity through a G-protein coupled receptor.


Key Words: T cell • multiple sclerosis • pertussis toxin • cAMP • caspase-3 • immune reconstitution syndrome

CEREBRAL ATROPHY and neuronal injury are important correlates of long-term disability in patients with chronic inflammatory diseases such as multiple sclerosis (MS). Although the mechanisms of neuronal injury are not well understood, several lines of evidence suggest that inflammatory infiltrates may be key factors in mediating cerebral atrophy and black holes or axonal transaction in patients with MS. For example, neuroimaging studies suggest that ring-enhancing patterns on MRI contribute to severe brain atrophy in patients with MS (1) . Several studies have shown that patients exhibit significant brain atrophy in the earliest stages of MS and that central nervous system (CNS) atrophy and axonal loss may develop at a faster rate in the first few years of disease onset (2 3 4 5 6 7) . Other reports have suggested that the rate of progression of CNS atrophy may be greater in more advanced relapsing–remitting (RR) phases of disease (8 9 10) . Further, treatment with pulse methylprednisolone (9) , blockade of cell migration with anti-VLA-4 monoclonal antibody (mAb) (11) or modulation of T cell phenotype with glatiramer acetate (12) resulted in less black hole formation on T-1 MR images and less loss in cerebral volume. These studies support the role of lymphocytic infiltrates in mediating neuronal damage. Further, pathological studies from MS patients have also shown that axonal injury may occur early during lesion evolution and is not related to demyelinating activity, which thus suggests that axonal injury is an independent process (13 14 15) . There is also evidence for apoptotic loss of neurons in the cerebral cortex (16) . Other neuroinflammatory diseases associated with T cell infiltrates in the brain such as Rasmussen’s encephalitis (17) and immune reconstitution syndrome in patients with HIV infection (18) are also associated with neuronal injury but have not been as well studied.

Immune abnormalities in MS include activated T cells in the blood and cerebrospinal fluid. T cells reactive to myelin components are found in a state of heightened activation and differentiation in patients with MS but not controls (19 20 21) . Although it is widely hypothesized that autoreactive T-cells recognize myelin proteins and initiate the inflammatory cascade, recent reports also indicated that activated CD8 T cells also induce axonal damage in vivo (22 , 23) . Further activated T cells can also cause direct neuronal toxicity in vitro through a contact-dependent mechanism (24) . But whether activated T cells induce neuronal toxicity through released soluble factors is still unclear. MRI studies suggest there is axon damage in normal appearing white matter and histopathological reports document low concentration neuronal and axonal damage in areas devoid of inflammatory cells, which suggest the possibility that soluble cytotoxic mediators may cause distant damage in MS tissues (23) . Further, GrB staining has been seen in MS brain tissue and was recently implicated as being as important mediator of tissue damage in progressive MS (25 , 26) and in other neuroinflammatory disorders such as Rasmussen’s encephalitis (27) .

Although the specific mechanism underlying T cell-induced direct neuronal toxicity is unknown, an important mechanism used by cytotoxic T cells to induce target cell death is through the granule exocytosis pathway involving the delivery of lymphocyte granule toxins to target cells (28) . Two major constituents of the lymphocyte granules are serine protease granzymes and the membrane-disrupting protein perforin. Among many members of the granzymes, granzyme B (GrB) is the best-characterized because of its strong proapoptotic activities. GrB is a 32-kDa serine protease and cleaves its substrates on the carboxyl side of acidic residues, especially aspartate. Although perforin made pores in the plasma membrane, originally thought to be necessary for GrB’s entry into the target cell, it has been shown that GrB can be taken up by target cells independent of perforin (29) . The uptake of GrB into the target cell has been shown to be partly mediated through the mannose-6-phosphate receptor (MPR). However, MPR is not an exclusive mechanism for GrB uptake, as MPR-deficient cells remained as vulnerable to GrB similar to wild-type cells (30) .

GrB signaling pathways involve activating caspases, a large family of endogenous cytosolic proteases that mediate apoptosis (31) . GrB also regulates mitochondrial outer membrane permeabilization via the cytosolic, proapoptotic protein Bid (32) . It has been shown that GrB cleaved Bid translocates to the mitochondria and interacts with other proapoptotic proteins such as Bax and Bak to induce release of cytochrome c from the mitochondria (33) . A caspase-independent pathway of GrB-induced apoptosis has also been identified in various cell lines exposed to GrB and perforin in the presence of a caspase inhibitor, z-VAD-fmk, when cell death could still proceed even though the nuclear apoptotic changes were largely abrogated (34) .

In the present study, we use human fetal primary neuronal cultures to demonstrate that activated T cells release soluble factors to induce neuronal toxicity. GrB is a critical factor in mediating T cell-induced neuronal toxicity and this appears to occur through a surface receptor-mediated pathway rather than the perforin dependent formation of membrane pores that is commonly cited. The mechanism and possible therapeutic agents against GrB-induced neuronal toxicity are also examined.

MATERIALS AND METHODS

Cells and cell cultures
Peripheral blood mononuclear cells were isolated from three different healthy human donors by standard ficoll separation from heparinized whole blood. CD4+ and CD8+ cells were isolated by negative selection using MACS beads (Miltenyi Biotec, Gladbach, Germany). T cell subsets were incubated at 37°C in Iscove’s modified Dulbecco’s medium supplemented with 5% human serum and activated (Ac) by placing on plates coated with 1 µg/ml of anti-CD3 and 1 µg/ml of soluble anti-CD28 for 72 h in culture. Culture supernatants were then collected and incubated (1:10 dilution) with human fetal neurons.

Human fetal neurons were cultured as described previously (35) . Briefly, human fetal brain specimens of 12–17 wk gestation were obtained in accordance with National Institutes of Health guidelines. The tissues were then triturated after removing the meninges. Cells were then cultured in T75 flasks in opti-MEM with 5% FBS, 0.5% N2 supplement and 1% antibiotics. Neurons were collected by carefully shaking the flask at least 1 month later. Cells were then seeded at 1 x 105/ml in 96-well plates for 1 wk before treatment. These cultures contain 70–80% neurons, <5% microglia, and the remaining cells are astrocytes as determined by immunostaining for microtubule-associated antigen (MAP-2), CD68, and glial fibrillary acidic protein (GFAP), respectively.

Enriched human fetal astroglia were cultured as described previously. Briefly, human fetal brain specimens of 12- to 17-wk gestation were triturated after removal of the meninges. Cells were then cultured in T75 flasks in Dulbecco’s modified Eagle medium with 10% FBS and 1% antibiotics for at least 1 month. After shaking at 180 rpm for 1 h, cells were separated with trypsin/EDTA. Cells were then seeded at 1 x 105/ml in 96-well plates for 1 wk before treatment. >95% of these cells were positively immunostained for GFAP.

Detection of GrB
To determine whether activated T cells release GrB extracellularly, T cell culture supernatants were collected and clarified by centrifuging at 9000 rpm for 10 min. The pellet was discarded. GrB in the supernatants was then determined by Western blot analysis. For Western blot analysis, 50 µl of the supernatant was concentrated by precipitation with tricholoroacetic acid. The pellet was then mixed with SDS sample buffer and boiled for 5 min. Samples were resolved on a 15% Tris-glycine polyacrylamide gel. After transfer of proteins to a polyvinylidene difluoride (PVDF) membrane, the blot was probed with a mAb to GrB. Immunoreactive bands were visualized by electrochemiluminescence (Amersham-Pharmacia Biotech, Piscataway, NJ). The intensity of the signal was quantified using a densitometer.

Immunodepletion of GrB
To immunodeplete GrB from T cell supernatants, the supernatants were incubated 1:1 with preswollen protein G sepharose (Amersham-Pharmacia) for 2 h at 4°C, a step taken to eliminate proteins in the lysate, which may bind nonspecifically to the protein G. The mix was subsequently spun, and the supernatant was incubated at 4°C overnight with anti-GrB or an isotype-matched control antibody (Ab). This mix was incubated for 2 h with protein G sepharose and filtered through a column. The supernatant was then used to treat neuronal cultures. All incubations (Ab and protein G) were performed on a rotary table at 4°C, and all centrifugations were performed using a desktop Eppendorf centrifuge at 4°C for 5 min at maximum speed (9000 g).

Neurotoxicity assays
Neurotoxicity was evaluated by using MTT and trypan blue uptake assays. For MTT assay, collected neurons were cultured at 1 x 105/ml in Locke’s buffer in 96-well plates. T cells culture supernatants (1:10 to 1:100 dilution) were then added and cultured for 44 h. MTT (5 mg/ml) was added to the cultures, and cells were incubated for another 4 h. Dimethyl sulfoxide (DMSO; 50%) was added to dissolve the formazan, and the optical density value was detected at 590 nM.

For trypan blue uptake, neuronal cells were seeded at 1 x 105/ml and incubated for 1 wk in 96-well plates before treatment. After adding the reagents (GrB (0.5–4 nM; Calbiochem, San Diego, CA), perforin (50 ng/ml), MnTMPyP (superoxide dismutase (SOD)/catalase mimetic; 10 µM; Calbiochem), pertussis toxin (PTX)(100 ng/ml; Calbiochem), Z-VAD-fmk (10 µM; BIOMOL, Plymouth Meeting, PA), D-mannose 6-phosphate (1 mM; Calbiochem), stromal-derived factor 1-{alpha} (SDF-1{alpha};), trolox (analog of vitamin E; 10 µM; Sigma, St. Louis, MO), immunophilin 3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrollidinecarboxylate (10 µM; GPI-1046; given by Guilford Pharmaceuticals, Baltimore, MD), the cells were incubated for another 48 h. The cells were then stained with trypan blue for 5 min, washed with PBS, pH 7.4 (PBS), and fixed with 4% paraformaldehyde. Trypan blue-positive and -negative cells were counted in three predetermined fields. Approximately 200 cells were counted in each well. Each experiment was done in triplicate wells, and mean and SEM were calculated from at least 3 independent experiments.

Caspase-3 activation
Human fetal neuron cultures on cover slips were treated with GrB (1 nM) for 24 h. Caspase-3 and ßIII tubulin expression in neurons was determined by immunocytochemistry. Briefly, cells were fixed with 4% paraformaldehyde for 5 min, and then blocked with 3% FBS in PBS for 20 min. Polyclonal caspase-3 antiserum (1:500) and monoclonal anti-ßIII tubulin (1 µg/ml; Promega, Madison. WI) Ab were then applied and cover slips incubated overnight at 4°C. After washing with PBS, the cover slips were incubated with secondary antibodies (1: 200 Donkey anti-rabbit IgG Alexa Fluor 594 and antimouse IgG Alexa Fluor 488, Molecular Probes, Eugene, OR) for 2 h. Hoechst 33258 (10 µM) was added at the last half hour to stain the nucleus. The cells were imaged by confocal microscopy.

Cyclic AMP assay
Human fetal neuronal cultures were seeded at 5 x 105/ml in 12-well plates for 1 wk before treatment. Cultures were then treated with GrB (1 nM) in Locke’s buffer for 0–30 min. After removing the media, we treated the cells with 100 µl of 0.1 M HCl for 10 min to achieve cell lysis. The lysate was centrifuged at 600 g for 10 min, and the supernatant was used directly for the cyclic AMP assay. Cyclic AMP competitive ELISA kit (ENDOGEN, Rockford, IL) was used according to manufacturer’s directions.

Calcium imaging
Cytosolic calcium ([Ca2+]c) was determined using the ratiometric calcium probe fura-2/acetoxymethyl ester using methods similar to those previously described (36) . Cells were incubated in 2 µM fura-2/acetoxymethyl ester for 20 min at 37°C in media and washed with Locke’s buffer (154 mM NaCl, 3.6 mM NaHCO3, 5.6 mM KCl, 1 mM MgCl2, 5 mM HEPES, 2.3 mM CaCl2, 10 mM glucose; pH 7.4) to remove extracellular fura-2/acetoxymethyl ester. Fura-2 loaded cells were placed into a open bath chamber and maintained at 37°C (Series 20 open perfusion chamber and TC-344B temperature controller; Warner Instruments. Hamden, CT). Buffer flowed over the cells at the rate of ~ 2 ml/min using a VC-6 perfusion control system with a multi-input manifold that minimized dead space, allowing for a rapid change between buffer and buffer containing drug. Cells were alternatively excited at 340 and 380 nM by a monochrometer, and emission was recorded at 510 with software from Intracellular Imaging (Intracellular Imaging, Cincinnati, OH.). 340/380 nM ratios were converted to nM [Ca2+]c using curve fitting software and calcium reference standards (Molecular Probes, Carlsbad, CA).

RESULTS

Activated T cells release GrB, which is neurotoxic
To determine whether activated T cells release neurotoxic soluble factors, we exposed cultured human fetal neurons to supernatants from purified T cells that had been activated with anti-CD3 and anti-CD28 antibodies and assessed neuronal viability by either a MTT assay or by trypan blue exclusion. We found that the culture supernatants from activated T cells induced significant toxicity to neurons compared to unstimulated T cells, and the toxicity was more prominent with supernatants from activated CD8+ cells (data not shown). These observations indicate that activated T-cells may release soluble factors to induce neuronal toxicity. Because GrB is an important factor in mediating T cell-induced cytotoxicity, we examined the production of GrB in the cultured T cell supernatants by semiquantitative Western blot analysis and ELISA. As shown in Fig. 1 A, all the supernatants contained GrB. However, activation of both CD4+ and CD8+ T cells increased release of GrB significantly compared to the corresponding controls (P<0.05). To further determine whether the released GrB was responsible for the neurotoxicity, we immunodepleted the GrB from the supernatants and found that neurotoxicity of the supernatants was significantly attenuated; however, when the supernatants were similarly treated with an isotype control Ab, no loss of neurotoxicity was noted, clearly demonstrating that the neurotoxicity of the T cell supernatants was, at least in part, due to GrB. (Fig 1B ).


Figure 1
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  		Figure 1. Activated T cells mediate neurotoxicity via GrB. Peripheral blood mononuclear cells (PBMC) or sorted CD4+ or CD8+ cells were cultured for 3 days in Iscove’s modified Dulbecco’s Medium +5% human serum. In parallel, these cells were incubated with anti-CD3/CD28 (cells:beads, 10:1) mAb-conjugated magnetic beads to induce polyclonal activation (Ac). Supernatants (sups) were then collected for GrB detection. Both activated CD4+ and CD8+ T cells released significant high amounts of GrB into the sups as determined by GrB immunoblot (A). Cultured human fetal neurons were treated with culture supernatants (1:10 in Locke’s buffer) from unsorted human T cells. Control cultures were treated with supernatants from unactivated T cells. To determine the role of GrB, activated T cell supernatant (AcT) was firstly immunodepleted with Ab against GrB (GrB-Ab) or control mouse IgG bound to protein A beads before treatment. Neurotoxicity was determined by calculating the percentage of cells positive with trypan blue 48 h later (B). Data represent the mean ± SEM of 3 replicates from 3 independent experiments. Image representative for 3 independent experiments is shown.

Recombinant GrB induces toxicity in neurons but not in astroglia
To further confirm that GrB could induce neurotoxicity, we used recombinant GrB (0.5 to 4 nM) and found that 1 nM was the minimum concentration needed to cause significant neurotoxicity as demonstrated by caspase-3 activation and nuclear fragmentation, suggestive of apoptosis (Fig. 2 ). We next determined the effect of perforin on GrB-induced neurotoxicity. We used threshold toxic concentrations of perforin (50 ng/ml) for these experiments. Some enhancement of GrB neurotoxicity may be present, but the enhancement was not statistically significant (P>0.05). Clearly, no synergistic effects were seen with GrB and perforin (Fig 3 A). These observations suggest that GrB alone may be sufficient to cause neurotoxicity. We next determined whether the GrB-mediated toxicity could also occur in astrocytes. Human astrocyte cultures were similarly treated with recombinant GrB (dose: 1–4 nM) and monitored for neurotoxicity. No evidence of cell death was noted in these cultures (data not shown). This suggests that GrB toxicity is specific for a subpopulation of neurons.


Figure 2
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  		Figure 2. GrB causes caspase-3 activation in neurons. A) Untreated neurons show immunostaining for ßIII tubulin (red) demonstrating normal morphology but are negative for caspase-3. B–F) GrB treatment-induced caspase-3 expression and apoptosis in some neurons. B) a ßIII tubulin-positive neuron shows a fragment nucleus, indicating apoptosis. C–F) a ßIII tubulin-positive (C) neuron shows increased expression of caspase-3 (D) and a fragmented nucleus (E).


Figure 3
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  		Figure 3. GrB-induced neurotoxicity is perforin and M6P receptor independent but mediated by Gi{alpha} receptors and caspase dependent pathways. A) Human neuronal cultures were treated with GrB (1nM), human perforin (50 ng/ml) or with the combination of the two. B) Human fetal neurons were treated with GrB in Locke’s buffer for 48 h. Mannose-6-phosphate (M6P, 1mM), pertussis toxin (PTX, 100 ng/ml), or caspase inhibitor Z-VAD-FMK (Z-VAD, 10 µM) were added 1 h before GrB treatment. Neurotoxicity was determined with trypan blue uptake assay. C) cAMP levels were measured in neuronal cultures after GrB treatment. GrB treatment results in a significant decreases (P<0.01) in cAMP concentration at 5 min. Data represent the mean ± SEM of 3 replicates from 3 experiments.

GrB-induced neurotoxicity is independent of mannose-6 phosphate receptor but is mediated by Gi{alpha}/Go coupled receptors and caspase-dependent pathways
Because GrB may enter cells in a perforin-independent manner via interactions with the mannose-6-phosphate receptor (37) , we pretreated the cells with 10 mM mannose-6-phosphate followed by GrB (1 nM). Mannose-6-phosphate was unable to inhibit GrB-induced neurotoxicity, suggesting that GrB-mediated neurotoxicity is independent of both perforin and mannose-6-phosphate receptor (Fig 3B ). However, GrB-mediated neurotoxicity could be significantly (P<0.05) blocked by Z-VAD-fmk (Fig 3B ), a broad spectrum caspase inhibitor, suggesting that GrB induces neuronal toxicity via activation of caspase-dependent apoptotic pathways. Interestingly, GrB-induced neurotoxicity could also be blocked by 10 µM pertussis toxin (PTX) (Fig. 3B ), suggesting a role for Gi{alpha}/Go-coupled receptors in apoptotic pathway-mediated neuronal cell death. Consistent with its ability to act on PTX-sensitive receptors, GrB stimulates a decrease in cAMP. cAMP levels were measured in neuronal cultures after GrB treatment. Decreases in cAMP levels occurred in a time-dependent manner with significant decreases at 5 min (Fig 3C ).

GrB disrupts calcium homeostasis in neurons
To determine whether GrB could disrupt calcium homeostasis, purified GrB was applied onto neurons and [Ca2+]c was measured in real time. A dose-dependent increase in the basal concentration of [Ca2+]c was noted within 30 min (Fig. 4 A). At the lowest concentration tested, 1 nM, GrB doubled the resting concentration of [Ca2+]c. Higher doses of GrB increased resting [Ca2+]c fourfold (Fig 4A ). Because elevated [Ca2+]c levels can result in endoplasmic reticulum (ER), calcium overload, and neuronal death, we determined whether GrB enhanced IP3-mediated ER calcium release using, SDF-1{alpha}, a G-protein-coupled receptor that stimulates ER calcium release via the Gi ß{gamma} subunits (38) . We found that pretreatment with GrB resulted in a marked increase of SDF-1{alpha}-evoked ER calcium release from a peak increase of 250 nM in vehicle-treated cultures to 1000 nM in cultures pretreated with GrB (Fig 4B ). The ability of GrB to potentiate the response of SDF-1{alpha} is consistent with its ability to stimulate a Gi protein-coupled receptor.


Figure 4
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  		Figure 4. GrB disrupts calcium homeostasis in neurons. Primary neurons were exposed to GrB for 30 min before imaging of intracellular calcium using fura-2AM. A) Resting levels of cytosolic calcium were increased by treatment with 1.0–100 nM GrB. B) SDF-1{alpha} (10 µM)-evoked increases of cytosolic calcium were increased by pretreatment of neurons with 10 nM GrB. *P < 0.001.

Attenuation of GrB-induced neurotoxicity with SOD/catalase mimetic, vitamin E, and neuroimmunophilin
To screen for possible agents that could protect against GrB-induced neurotoxicity, we pretreated the cultures with 10 µM MnTMPyP, a SOD/catalase mimetic, and we found that it significantly blocked the neurotoxicity (Fig 5 A, P<0.05). Similarly, we found that 10 µM trolox, analog of vitamin E, and neuroimmunophilin GPI-1046 also blocked GrB neurotoxicity (P<0.05) (Fig 5B ). GPI-1046 is an agent with both neuroprotective and neurotrophic effects, but the exact mechanism of action remains unknown.


Figure 5
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  		Figure 5. Antioxidants and GPI compounds protect neurons from GrB-induced toxicity. Neuronal cultures were treated with GrB in Locke’s buffer in the presence or absence of SOD/catalase mimetic MnTMPyP (MnT, 10 µM, A), Trolox (Vitamin E, 10 µM), GPI-1046 (10 µM), which were added 1 h before GrB treatment. Neurotoxicity was then determined 48 h later with trypan blue uptake assay. Data are shown as the mean ± SEM of 3 replicates from 1 experiment, representing 3 independent experiments with similar results. *P < 0.05, compared to control; #P < 0.05, compared to GrB alone-treated groups.

DISCUSSION

It is well accepted that T cell activation plays an important role in the pathogenesis of MS. There is a significant correlation between the extent of axon damage and the numbers of T cells in MS plaques (39) . Activated T cells can induce direct neuronal damage via cell-to-cell contact (24) . In the present study, we found that activated T cells could also induce neuronal toxicity in a cell contact-independent manner by releasing soluble toxic factors. Among these soluble factors, GrB plays an important role in mediating neuronal toxicity. GrB is stored in granules in restive T cells along with other cytotoxic factors, including perforin. On activation, T cells release GrB from the granules to act on target cells. Interestingly, we observed that both activated CD4- and CD8-positive T cells release GrB into the supernatant in our culture system. This observation is consistent with reports that besides CD8 cells, subsets of CD4-positive cells can also express GrB (40 , 41) . The basal concentration of GrB release from the T cells is likely due to partial activation of T cells in culture. We found that the toxicity of GrB was specific for neurons, whereas no toxicity was observed in astrocytes. This is also consistent with a previous study that showed that astrocytes and oligodendrocytes are resistant to toxicity by cytotoxic T cells (24) .

GrB is a serine protease that induces apoptosis by caspase activation after crossing the plasma membrane of target cells. In several systems/cell types, the ability of GrB to enter the target cells is dependent on perforin (42) . In other systems, GrB entry is facilitated by expression of the mannose-6-phosphate receptor (37) . We demonstrate that perforin played a minimal role, if any, in GrB-mediated neurotoxicity, and the effect was independent of the mannose-6-phosphate receptor. This is consistent with recent observations that GrB binding to the cell membrane is cell type specific, and the toxicity in these cells can be independent of both perforin and mannose-6-phosphate (43) .

Our observations further suggest that GrB-mediated effects were PTX sensitive. This implicates the stimulation of the Gi{alpha}-coupled receptors (44) . Conceivably, these interactions may occur at the cell membrane and may not require GrB uptake. Alternatively, GrB may generate a ligand by cleavage of extracellular matrix that then interacts with the Gi{alpha} coupled receptors. Moreover, it is becoming increasingly recognized that a number of cell surface receptors may be cleaved by proteinases with a consequent change in the activity of intracellular signaling molecules. For example, granzyme A may, like thrombin, activate G protein-coupled class of proteinase-activated receptors (45) . Although some studies suggest that GrB does not influence thrombin signaling in a manner that would be expected, thrombin-insensitive proteinase-activated receptors are known to occur (46) .

Gi{alpha}-coupled receptors are known to regulate adenylyl cyclase, which, in turn, regulates cAMP levels. However, adenylyl cyclase is differentially regulated by the Gi family of heterotrimeric G-proteins and is stimulus dependent (47) . Hence, we measured cAMP levels in the neuronal cultures after GrB treatment and found that a decrease in cAMP levels. This may have important implications for neuronal function, since as a second messenger, cAMP plays a critical role in multiple forms of synaptic plasticity and in long-term memory formation (48 , 49) .

We found that GrB disrupted calcium homeostasis by increasing resting levels of [Ca2+]c and enhancing IP3-mediated ER calcium release to attain concentrations of [Ca2+]c that are sufficient to activate calcium-dependent death effectors, including the caspases. Because SDF-1{alpha} levels are elevated in the CSF of patients with neuroinflammatory disorders (50) , in areas of inflammation, GrB-mediated ER calcium overload may result in a synergistic and lethal response to SDF-1{alpha} suggesting that, even physiological or slightly elevated levels of such chemokines in the appropriate setting may be sufficient to cause neuronal injury and death.

We also found that GrB-mediated neuronal cell death involved caspase activation and could be prevented by antioxidants such as MnTMPyP and vitamin E, suggesting an important role for oxidative stress upstream of caspase activation. This may have important therapeutic implications. Several pathological studies from MS patients also suggest that a significant amount of oxidative stress occurs in chronic active plaques (51) and plays a role in neurodegeneration (52) . However, most of the antioxidant agents do not readily cross the blood-brain barrier. The neuroimmunophilins are hence of special interest, since they have substantial antioxidative properties and favorable pharmacokinetic profiles that include good CNS penetration.

In conclusion, we show that activated T cells release GrB which causes neurotoxicity via novel membrane-mediated interactions. GrB causes stimulation of Gi{alpha} receptors, leading to a decrease in cAMP levels, increase in intracellular calcium- induction of oxidative stress and activation of caspases (as depicted in Fig. 6 ). These observations may have important implications for T cell-mediated neuroinflammatory diseases such as MS. Further this model system will be useful to dissect the molecular mechanisms of T cell mediated neuronal death and offers the potential for high throughput screening of neuroprotective compounds.


Figure 6
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  		Figure 6. Schematic flow chart depicting the possible mechanism for GrB-mediated activated T cells supernatant-induced neuronal damage. CD3/CD28 activated T cells release GrB into the supernatants. GrB induces neurotoxicity by both perforin-dependent or -independent mechanisms. As for the latter, GrB activates PTX-sensitive Gi-coupled receptors by either direct binding, or by cleaved fragments binding or by direct cleavage. The activation of Gi-coupled receptor then results in decreased cAMP levels and elevated intracellular calcium levels, which may induce caspase-3 activation, leading to neuronal apoptosis. It is likely that oxidative stress also participates in the neurotoxicity, and it is upstream of caspase activation.

ACKNOWLEDGMENTS

This study was supported by Grants P01MH070056, R01NS039253, and R01NS043990 from the National Institutes of Health and a cooperative grant from the National Multiple Sclerosis Society.

Received for publication November 29, 2005. Accepted for publication January 20, 2006.

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