Brain, Vol. 125, No. 11, 2469-2480,
November 2002
© 2002 Oxford University Press
TRAIL induces death of human oligodendrocytes isolated from adult brain
1 Departments of Neurology and 2 Neurosurgery, Medical University of Lodz, Poland
Correspondence to: Krzysztof Selmaj, MD, PhD, Department of Neurology, Medical University of Lodz, 22 Kopcinskiego Street, 90-153 Lodz, Poland E-mail: kselmaj{at}afazja.am.lodz.pl
Received June 2, 2002. Accepted June 16, 2002.
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Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to induce apoptosis in various tumour cell lines and, recently, also in normal cells. TRAIL interacts with four receptors: two signalling receptors (TRAIL-R1 and TRAIL-R2) and two decoy receptors (TRAIL-R3 and TRAIL-R4). We have shown that both signalling receptors are present on the surface of oligodendrocytes isolated from adult human brain (ahOL), whereas the decoy receptors are expressed at a low level on ahOL. TRAIL induces ahOL apoptosis—as characterized by Annexin V staining prior to propidium iodide cell uptake—under conditions of protein synthesis inhibition. However, pre-treatment of ahOL with interferon
(IFN) evoked susceptibility to TRAIL-induced death, which did not require inhibition of protein synthesis. A blocking experiment with monoclonal antibodies directed against TRAIL-R1 and TRAIL-R2 revealed that TRAIL-R1 is mainly involved in TRAIL-induced apoptosis of ahOL. In contrast to ahOL, microglial cells were completely resistant to cell death induced by TRAIL. Microglial cells had high surface expression of the decoy receptor TRAIL-R3, suggesting that resistance of these glial cells to TRAIL-induced death depends on the presence of the protective effect of TRAIL-R3. Stimulation of microglia with TRAIL increased further expression of TRAIL-R3, but it had no effect on the expression of TRAIL receptors by ahOL. This result may implicate TRAIL as an effector-immune molecule in selective ahOL demise in inflammatory/demyelinating conditions. Keywords: TRAIL; oligodendrocytes; glial cells; death receptor; brain
Abbreviations: ahOL= adult human oligodendrocytes; CHX = cycloheximide; FITC = fluoroisothiocyanate; IFN = interferon; PI = propidium iodide; TNF = tumour necrosis factor; TRAIL = tumour necrosis factor-related apoptosis-inducing ligand; TRAIL-LZ = TRAIL leucine-zipper; TRAIL-R = TRAIL receptor
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Introduction |
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Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors form a potent ligand–receptor system, which is responsible for cell death. TRAIL is homologous to other death-signalling molecule family members such as Fas-L, tumour necrosis factor (TNF) and lymphotoxin (LT)-
and LTß (Pitti et al., 1996
). The unique feature originally attributed to TRAIL was selective TRAIL-induced apoptosis of tumourigenic or transformed cells, but not normal cells (Willey et al., 1995). More recently, however, it has been shown that TRAIL can induce apoptosis of normal hepatocytes, indicating that TRAIL-induced cell death is not restricted to transformed cells (Jo et al., 2000). These results widened the potential pathogenic role of TRAIL to other conditions involving cell death such as inflammation and autoimmunity. TRAIL is expressed widely in many cell types and tissues, and the regulation of TRAIL-induced death is through restricted expression of its receptors (Pan et al., 1997). Four TRAIL receptors (TRAIL-Rs) have been cloned: TRAIL-R1 and TRAIL-R2 are functionally active receptors, whereas TRAIL-R3 and TRAIL-R4 are non-signalling decoy receptors. The mutual expression of signalling and decoy receptors determines whether cells are sensitive or resistant to TRAIL-induced death (Degli-Esposti et al., 1997; Sheridan et al., 1997; Walczak et al., 1997). However, the hypothesis is not entirely accurate because there is no obvious correlation between the mRNA for TRAIL-R3 and TRAIL-R4 and resistance to TRAIL-induced death in many tumour cell lines (Griffith et al., 1998).
Little is known about TRAIL-induced effects in the CNS. Several CNS-related conditions involve neuronal and glial cell death. Recently, Nitsch and colleagues (Nitsch et al., 2000) used a brain slice culture system to show that TRAIL induced extensive and non-selective brain cell death. These results may indicate particular sensitivity of CNS cells to TRAIL. Depletion of oligodendrocytes is a recognized feature of multiple sclerosis lesions. Several immune mechanisms have been proposed to induce oligodendrocyte death including non-major histocompatibility complex (MHC) restricted injury by other TNF family members such as Fas-L, TNF and LT (Raine, 1994; Brosnan and Raine, 1996; Selmaj and Raine, 1998). These results indicate that death receptor ligation may provide a major destructive signal to oligodendrocytes in autoimmune inflammatory lesions.
In this study, we have assessed (i) the expression of TRAIL-Rs on oligodendrocytes isolated from adult human brain (ahOL) and microglial cells; and (ii) the effect of TRAIL on ahOL and microglial cell survival.
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Reagents
TRAIL leucine-zipper (TRAIL-LZ) and mouse monoclonal antibodies against human TRAIL(M180), TRAIL-R1(M271), TRAIL-R2(M413), TRAIL-R3(M430), TRAIL-R4(M444) were a generous gift from Immunex Corporation (Seattle, WA, USA). The antibodies’ specificity for different TRAIL-Rs has been described previously (Griffith et al., 1999
). Briefly, antibodies against TRAIL-Rs were generated after immunization of mice with fusion proteins that consisted of the extracellular fraction of TRAIL-Rs linked to a constant region of human IgG1. Specificity of these antibodies was confirmed by flow cytometry and western blot analysis performed on CV-1 cells transfected with cDNA encoding the whole sequence of each receptor. No cross-reactivity could be detected in these assays.
Anti-mouse horseradish peroxidase (HRP) antibody (Santa Cruz, CA, USA), anti-mouse fluoroisothiocyanate (FITC) conjugate antibody and propidium iodide (PI) were obtained from Sigma (Poznan, Poland). Annexin V–FITC was purchased from Pharmingen (San Diego, CA, USA), interferon (IFN) from R&D Systems (Minneapolis, MN, USA), TNF from Endogen (Boston, CT, USA) and the human cDNA library from Clontech (Franklin Lakes, NJ, USA).
Target cells
Adult human oligodendrocytes (ahOL) obtained from neurosurgical procedures were prepared from adult human brain resected as a surgical treatment for tumours as described previously (Jurewicz et al., 1998). Briefly, the tissue was treated with trypsin, rubbed through a mesh and centrifuged on a 30% Percoll gradient. The dissociated cells were suspended in minimum essential medium with 5% foetal calf serum (FCS), streptomycin (50 µg/ml) and penicillin (50 U/ml) (all from Gibco BRL, Life Technologies, Paisley, UK) before being cultured for 48 h in a culture flask. This step enabled adherent cells such as microglia and astrocytes to be separated from non-adherent cells such as oligodendrocytes. The non-adherent oligodendrocyte fraction was plated at a concentration of 5 x 104 cells/well onto 96-well microtitre plates coated with poly-L-lysine and cultured for 2 weeks. The purity of these cultures was 
90% (Jurewicz et al., 1998). After purification, the cells (ahOL and microglia) were kept in culture for the same period of time before use in the experiments.
A cell line (MO3.13) formed by the fusion of ahOL and a rhabdomyosarcoma cell line (a generous gift from Dr Neil Cashman) was cultured in high glucose Dulbecco-modified Eagle’s medium supplemented with 10% FCS, 2.5 U/ml penicillin, 2.5 µg/ml streptomycin and 2 mM L-glutamine at 37°C. A T98G glioma cell line was cultured under the same conditions. HELA and Jurkat cell lines were cultured in RPMI (Roswell Park Memorial Institute medium) with 10% FCS, 2.5 U/ml penicillin and 2.5 µg/ml streptomycin.
Before the experiments, all cell lines were seeded on 24-well plates, left for 24–48 h and then stimulated with TRAIL. ahOL were stimulated with TRAIL in a serum-deprived condition, 24 h pre-treatment with cycloheximide (CHX) (10 µg/ml) or IFN (100 U/ml). All cells were stimulated with 300–2000 ng/ml TRAIL-LZ (Immunex).
In the blocking experiment, ahOL pre-treated with CHX were incubated with anti-TRAIL-R1 or/and anti-TRAIL-R2 monoclonal antibodies (10 µg/ml) for 1 h before exposure to TRAIL.
Annexin V–FITC conjugation and PI cell staining
Annexin V–FITC and PI were used to determine the percentage of cells undergoing apoptosis after exposure to TRAIL. After incubation with TRAIL for 5, 24, 48 and 72 h, the cells were washed twice with cold phosphate buffered saline (PBS) pH 7.4 and then resuspended in calcium-containing binding buffer (Pharmingen) at a concentration of 1 x 106 cells/ml. Next, 5 µl of FITC-conjugated Annexin V and PI (5 µg/ml) were added in a calcium-containing buffer (Pharmingen) to 100 µl of cell suspension. After 15 min incubation in the dark at room temperature, the cells were immediately analysed by flow cytometry (see below). As described by Vermes and colleages (Vermes et al., 1995), the apoptotic cells were defined as showing positive Annexin V staining prior to the appearance of PI staining.
Determination of surface expression by flow cytometry
Surface expression of TRAIL-Rs was determined by flow cytometric analysis by measuring the binding of anti-TRAIL-Rs monoclonal antibodies (M271, M413, M430, M444) (Immunex). Cells were incubated with TRAIL (300 ng/ml), TNF (1000 U/ml) and IFN (100 U/ml) for 24 h prior to analysing the expression of the receptors. Briefly, cells were incubated with monoclonal antibodies for TRAIL-Rs (3 µg/ml) for 1 h on ice, and then with anti-mouse FITC-conjugated antibody (Sigma) (1 : 100) for 30 min on ice (dilution with PBS). After several washes with PBS, cells were analysed using a fluorescence-activated cell sorter (FACS) (Becton Dickinson, San Jose, CA, USA).
Western blotting
Cells were lysed in TBS buffer (Tris buffered saline: 0.05 M Tris, 0.138 M NaCl, 0.0027 M KCl, pH 8.0) containing phenylmethylsulphonyl fluoride, aprotinin and Triton X-100 at 25°C. The lysates were centrifuged at 14 000 g to remove cellular debris. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to a polyvinylidene fluoride membrane (Millipore Corp., Bedford, MA, USA) and blocked with 5% non-fat dry milk (NFDM) in PBS–Tween-20 (0.05%) overnight at 4°C. The membrane was immunoblotted with monoclonal antibody directed against TRAIL-Rs (1 µg/ml in 5% NFDM in PBS–Tween-20) for 1 h as described previously (Griffith et al., 1999). After washing, the membrane was incubated for 1 h with an anti-mouse HRP antibody. Following several washes, the blots were developed by chemiluminescence with ECL Plus according to the manufacturer’s protocol (Amersham Pharmacia, Little Chalfont, UK).
Reverse transcription–polymerase chain reaction (RT–PCR) for TRAIL-Rs
Total RNA was isolated from cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA synthesis was performed using an oligo(dT) primer. Reverse transcription was performed using a thermal programme of 25°C for 10 min, 42°C for 30 min and 95°C for 5 min. PCR was performed using the following primers: ß-actin (forward: 5'-GAAACTACCTTC AACTTCCATC-3', reverse: 5'-CGAGGCCAGGATGGAGCCGCC-3'); TRAIL-R1 (forward: 5'-CTGAGCAACGCAGACTCGCTGTCCAC-3'; reverse: 5'-TCCAAGGACACG GCAGAGCCTGTGCCAT-3'); TRAIL-R2 (forward: 5'-GCCTCATGGACAATGAGATAAAGGTGGT-3', reverse: 5'-CCAAATCTCAAAGTACGCACAAACGG-3'); TRAIL-R3 (forward: 5'-GAAGAATTTGGTGCCAATGCCACTG-3', reverse: 5'-CTCTTGGACTTGGCTGGGAGATGTG-3'); TRAIL-R4 (forward: 5'-CTTTTCCGGCGGCGTTCATGTCCTTC, reverse: 5'-GTTTCTTCCAGGCTGCTTCCCTTTGTAG); TRAIL (forward: 5'-CAACTCCGTCAGCTC GTTAGAAAG-3', reverse: 5'-TTAGACCAACAACTATTTCTAGCACT-3').
Human ß-actin PCR cycle conditions were 95°C for 45 s, 55°C for 1 min, and 72°C for 45 s for 30 cycles. Human TR-1, TR-2 and TR-3 conditions were 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min for 30 cycles. Human TR-4 cycle conditions were 95°C for 4 min 15 s, followed by 30 cycles of 95°C for 45 s, 60°C for 45 s, and 72°C for 45 s (Griffith et al., 1998). Human TRAIL cycle conditions were 95°C for 45 s, 55°C for 45 s, and 72°C for 45 s for 30 cycles (Fanger et al., 1999). Samples were resolved on a 1% agarose gel and visualized with ethidium bromide.
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TRAIL induces ahOL apoptotic cell death
To determine whether oligodendrocytes and microglial cells are sensitive to TRAIL-LZ-induced apoptosis, we used Annexin V and PI staining, and cell analysis by flow cytometry to detect externalization of phosphatidylserine and PI uptake (indicating membrane disruption). When exposed to TRAIL-LZ at a concentration of 300–2000 ng/ml for up to 72 h, ahOL were resistant to cell death (Fig. 1A and B). Serum deprivation condition, which is known to increase cell susceptibility to other death receptor ligands (D’Souza et al., 1995
), did not decrease the survival of ahOL treated with TRAIL (data not shown). However, when ahOL were pre-treated with a protein synthesis inhibitor, CHX (10 µg/ml), for 24 h prior to TRAIL exposure, apoptosis was observed as detected by Annexin V staining preceding to PI cell uptake (Fig. 1A and B). Positive Annexin V–FITC staining of ahOL was observed 24 h after TRAIL exposure whereas PI uptake was detected only after 72 h. Cells that stained positively for Annexin V–FITC and negative for PI were considered to be undergoing apoptosis, whereas cells that stained positively for both Annexin V–FITC and PI were considered to be either in the end stage of apoptosis or already dead (Vermes et al., 1995). We continued ahOL observation for up to 120 h and did not see cell recovery, indicating that TRAIL-induced cell death and increased permeability for PI was irreversible. Regardless of protein synthesis inhibition, microglial cells were completely resistant to TRAIL-LZ for up to 72 h of incubation (Fig. 1A and B). As a control, we used three human cell lines representing transformed cells MO3.13, T98G and HELA, respectively. All these cell lines died within 24 h after TRAIL stimulation (Fig. 1A and B) and their death did not require inhibition of protein synthesis. This agrees with published results indicating high susceptibility of transformed cells to TRAIL-induced death (Kim et al., 2000). Since IFN was implicated in increasing cell death susceptibility to other TNF family ligands, we assessed whether pre-treatment of ahOL and microglia cells with IFN evoked cell death induced with TRAIL. Figure 1C shows that IFN added 24 h prior to TRAIL made ahOL susceptible to TRAIL-induced cell death. The susceptibility to ahOL death induced by IFN in response to TRAIL may be related to decreased expression of TRAIL-R3 (see below).
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Expression of TRAIL-Rs in glial cells
We used western blotting and flow cytometry to (i) determine the correlation between the expression of TRAIL-Rs in glial cells and their susceptibility to TRAIL-mediated cell death; and (ii) define their surface or intracellular localization. Using these two methods, we found that ahOL, microglia and the control lines MO.313, T98G and HELA all expressed TRAIL-R1 and TRAIL-R2 both on the cell surface and intracellularly (Fig. 2A and B). As previously reported (Sprick et al., 2000
), the Jurkat cell line expressed only TRAIL-R2 and a low level of TRAIL-R3 both on the cell surface and intracellulary. Interestingly, however, ahOL and microglial cells had a single band of immunoreactivity for TRAIL-R2 on the western blot compared with the double bands showed by most of the transformed control cell lines. This may indicate differences in the function of TRAIL-R2 between normal and transformed cells (see below). Microglial cells, which are resistant to TRAIL-induced death, expressed high levels of TRAIL-R3 on the cell surface, whereas ahOL expressed low levels of TRAIL-R3 and MO3.13, T98G and HELA cells did not express TRAIL-R3 (Fig. 2A). However, western blotting detected expression of TRAIL-R3 in all the studied cell populations (Fig. 2B). These results may indicate facilitated translocation of TRAIL-R3 from an intracellular compartment to the cell surface in microglia cells, which are resistant to TRAIL-induced death, but not in the other cells which are sensitive to TRAIL-induced death. The expression of TRAIL-R4 on the cell surface was absent from all the studied cell populations, but weak immunoreactivity was detected with western blotting (Fig. 2B).
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mRNA expression of TRAIL-Rs in glial cells
To further investigate the function of TRAIL-Rs in glial cells, we measured their transcription efficacy by assessing mRNA expression for TRAIL-R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4. Microglia cells, ahOL, HELA and Jurkat cells were strongly positive for TRAIL-R2 mRNA. In addition, expression of mRNA encoding TRAIL-R1 was detected in ahOL and microglial cells. TRAIL-R3 mRNA was expressed strongly in microglia; this correlated with its surface expression at a protein level in these cells (see above). Jurkat cells also expressed TRAIL-R3 mRNA. All investigated cells were negative for TRAIL-R4 mRNA, although the specificity of PCR reaction was confirmed with the human cDNA library (Fig. 3).
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TRAIL-induced ahOL death is mediated by TRAIL-R1
To assess which receptor is involved in the TRAIL-induced death of ahOL, we performed an experiment in which we added blocking antibodies against TRAIL-R1 and/or TRAIL-R2 for 1 h prior to TRAIL exposure. Then we determined cell death by staining the cells with Annexin V and PI. These experiments showed that blocking with TRAIL-R1 antibody protects ahOL from cell death whereas the antibody to TRAIL-R2 had no effect on TRAIL-induced apoptosis of ahOL (Fig. 4). There was also no synergistic effect when both anti-TRAIL-Rs were added. We used Jurkat cells, which expressed only TRAIL-R2, as a positive control of antibody against TRAIL-R2 (Sprick et al., 2000
). We observed a strong inhibitory effect of this antibody on the TRAIL-induced death of Jurkat cells (Fig. 4). These results indicate that the death signal in ahOL depends on the TRAIL–TRAIL-R1 interaction.
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Regulation of the expression of TRAIL-Rs in glial cells
Previous results have shown that cytokines can down-regulate or up-regulate TRAIL and TRAIL-R expression (Kayagaki et al., 1999
a, b; Sedger et al., 1999). To assess regulation of TRAIL-R expression in glial cells, we measured TRAIL-Rs on the surface of ahOL and microglia after stimulation with TNF and IFN; no increase in the expression of TRAIL-Rs was observed (data not shown). When TRAIL ligand was added, however, we observed strong up-regulation of TRAIL-R3 expression on microglia cells but not on ahOL cells (Fig. 5). These results indicate that TRAIL ligand can up-regulate TRAIL-R3 expression on microglia in an autoregulatory manner and protect the cells from death. Interestingly, adding IFN to ahOL decreased the already low expression of TRAIL-R3; these results correlated with the IFN-induced susceptibility to TRAIL-mediated death of ahOL (Fig. 5).
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In this study, we have shown that both the TRAIL cell death mediating receptors, TRAIL-R1 and TRAIL-R2, are expressed by ahOL isolated from human adult brain and that ligation of TRAIL-R1 induces ahOL death in the presence of protein synthesis inhibition or pre-treatment with IFN
. The susceptibility to TRAIL-induced death demonstrated by ahOL depends on low expression of decoy TRAIL-R3. This was in contrast to microglia cells, which expressed high levels of TRAIL-R3 and were resistant to TRAIL-induced death.
In other cell culture systems, TRAIL-induced death has also been shown to be evoked or enhanced by protein synthesis inhibitors such as CHX (Bretz et al., 1999) or actinomycin D (Griffith et al., 1998). This suggests that CHX and actinomycin D reduce the relative concentration of labile protein inhibitors in the TRAIL transduction pathway. The family of IAP (inhibitor of apoptosis protein) molecules has been shown to protect cell death induced by TNF family ligands; these molecules bind to and inhibit the activity of caspases—preferentially caspase-3 and caspase-9 (Ducket et al., 1996). Similarly, FLIP [FADD-like ICE (FLICE) inhibitory protein] inhibits the death receptor inducing signal by preventing caspase-8 and caspase-10 association with the adapter molecule FADD (Inohara et al., 1997; Irmler et al., 1997). It has recently been shown that ligation of TRAIL-R1 and TRAIL-R2 initiated FADD and caspase-8 association (Kischkel et al., 2000) and, accordingly, FLIP has been reported to inhibit TRAIL signalling (Wajant et al., 2000). In addition, the sensitivity of tumour cells to TRAIL was linked with the fact that they express low levels of FLIP (Kim et al., 2000). We have also detected expression of FLIP in ahOL and found that CHX decreased endogenous FLIP protein levels rapidly (data not shown). All these data indicate that TRAIL, despite being primarily toxic to transformed cells, can also mediate normal cell death—including that of ahOL—under appropriate conditions (mainly related to the inhibition of protein synthesis). Of particular importance is that susceptibility to TRAIL-induced death of ahOL can also be evoked by pre-treatment with IFN. The synergistic activity of IFN with TNF family ligands has been shown previously and was suggested to be related to the IFN-induced expression of TNF receptors (Pouly et al., 2000). In this study, however, we have shown that IFN-induced susceptibility of ahOL to TRAIL depends on the decreased expression of a decoy TRAIL-R3.
The observation that TRAIL can induce selective cell death of ahOL may be of importance to inflammatory/demyelinating conditions (including multiple sclerosis) in which the demise of oligodendrocytes is a recognized pathological feature. Thus, TRAIL can be added to the family of immune effector molecules that can be instrumental in ahOL demise. TRAIL, like TNF and FasL, has both membrane and trimeric soluble forms (Pitti et al., 1996). Although TRAIL mRNA has been found in various tissues and cells, both its expression at protein levels and its physiological function are still largely unknown. TRAIL expression by activated T lymphocytes and natural killer (NK) cells suggests that it may be involved in cell-mediated cytotoxicity (Kashii et al., 1999; Kayagaki et al., 1999c). TRAIL production has been shown to be under control of cytokines, e.g. type I interferons induced TRAIL expression on human T cells; IFN up-regulated TRAIL expression on murine liver NK cells; and interleukin-2 (IL-2) and IL-15 induced TRAIL expression on murine spleen NK cells (Kayagaki et al., 1999a, b; Sedger et al., 1999). Lipopolysaccharide increased TRAIL expression on human monocytes (Halaas et al., 2000). In addition, TRAIL was detected on autoimmune T cells (Wendling et al., 2000), which can use it as an effector molecule against CNS targets. Unlike soluble TNF receptors (Selmaj and Raine, 1995), however, a soluble TRAIL receptor inducing TRAIL blockade exacerbated experimental autoimmune encephalomyelitis (Hilliard et al., 2001). Interestingly, the CNS of mice treated with a soluble TRAIL receptor did not contain any more apoptotic cells, but lymphocyte production of cytokines in response to the encephalitogenic antigen, myelin oligodendrocyte glycoprotein (MOG), was increased (including both Th1 and Th2 types of cytokine). It was concluded that TRAIL might act differently to other TNF family ligands in experimental autoimmune encephalomyelitis and inhibit this disease. However, an alternative explanation may be that TRAIL inhibition prolonged survival of lymphocytes in the peripheral lymphatic organs leading to their prolonged activation and enhancement of experimental autoimmune encephalomyelitis. It is not clear whether the soluble TRAIL receptor construct was able to pass through the blood brain barrier, enter the brain and interfere with TRAIL’s interaction with oligodendrocytes.
Our results have clearly shown that TRAIL induced selective death of oligodendrocytes; this was underscored by the inability to kill microglia cells, a companion glial cell population. Recently, Nitsch and colleagues (Nitsch et al., 2000) reported that incubation of human brain slices with human recombinant TRAIL trimerized with a FLAG-specific antibody resulted in extensive brain cell death (as measured by nuclei staining with PI). PI-labelled cells in TRAIL-treated brain slices were identified by double-fluorescence immunocytochemistry as neurones, oligodendrocytes, astrocytes and microglial cells. These results indicated that TRAIL evoked a general destructive effect against neurones and glial cells in this brain slice system.
Extensive studies of the sensitivity of CNS cells to other members of the TNF family have shown great selectivity in their response to TNF family molecules. The cells that consistently demonstrated susceptibility to TNF and LT were oligodendrocytes, whereas astrocytes, microglial cells and neurones showed rather protective or proliferative responses (Selmaj et al., 1991; Selmaj and Raine, 1998). In contrast to the TRAIL killing of oligodendrocytes reported by Nitsch and colleagues (Nitsch et al., 2000), the kinetics of TRAIL-induced oligodendrocyte apoptosis in our experimental system with purified glial cell culture was similar to that reported previously for TNF and LT (Hisahara et al., 1997), where oligodendrocytes were killed 48–72 h post-exposure to TRAIL.
The differential sensitivity of a wide variety of cell types to TRAIL has been attributed to differences in the expression of TRAIL-Rs (Griffith and Lynch, 1998; Zhang et al., 2000a). TRAIL mediates apoptotic cell death by interaction with two distinct receptors containing death domains, TRAIL-R1 and TRAIL-R2 (Pan et al., 1997; Walczak et al., 1997). However, TRAIL-R1 and TRAIL-R2 are widely expressed on most cell types and, therefore, the alternative hypothesis is that the non-signalling TRAIL-Rs, TRAIL-R3 and TRAIL-R4, act as decoy receptors and determine whether a cell is resistant or sensitive to TRAIL-induced cell death (Degli-Esposti et al., 1997; Sheridan et al., 1997).
Our results showed that both TRAIL-R1 and TRAIL-R2 are expressed on oligodendrocytes and microglial cells to the same level. However, the blocking experiments demonstrated that TRAIL-R1 is responsible for mediating the death signal in ahOL. In most of the transformed cells, TRAIL-induced death is mediated by TRAIL-R2 (MacFarlane et al., 1997). The pattern of immunoreactivity of TRAIL-R2 in western blotting differed between ahOL and most of the control-transformed cell lines, which showed two bands instead of one band. This might suggest that, in normal cells, TRAIL-R2 is less prone to mediate the death signal and that TRAIL-R1 is the primary death mediating receptor of the TRAIL pathway. Such selective oligodendrocyte sensitivity to TRAIL may be related to a differential expression of the decoy receptors on these cells. Consistent with this hypothesis is our observation that microglial cells, which are resistant to TRAIL-induced cell death, expressed high levels of decoy TRAIL-R3 whereas oligodendrocytes and the transformed cell lines, MO3.13 and glioblastoma, expressed low levels of TRAIL-R3 or none at all. The differential expression of TRAIL-R3 was evident with flow cytometry, but not from the western blot analysis. This discrepancy could be explained by impaired translocation of TRAIL-R3 from the cytosol to the surface in oligodendrocytes and transformed cells. Decoy receptors are predominantly located within the cells in the nucleus, and their location within the cell suggested that expression of the receptors may involve regulated movement from intracellular compartments to the membrane (Zhang et al., 2000b). The translocation of decoy receptors to the cell surface depends upon a signal from TRAIL-R1 and TRAIL-R2. This was in our study in which TRAIL stimulation led to enhanced TRAIL-R3 expression by microglia. However, the translocation of TRAIL-R3 did not occur in ahOL. The mechanism of the failure to effectively translocate TRAIL-R3 to the surface of ahOL is not known, but could be responsible for ahOL sensitivity to TRAIL-induced death. In support of this notion, the levels of mRNA encoding TRAIL-R1 and TRAIL-R2 were equally high in ahOL and microglia, but the mRNA for TRAIL-R3 was much higher in microglia. This suggests increased metabolic turnover of this decoy receptor in cells resistant to TRAIL-induced death. The mRNA for the other decoy receptor, TRAIL-R4, was not detected in the studied cell populations. This was in agreement with previous reports showing low and inconsistent expression of mRNA for this receptor (Griffith et al., 1998).
In conclusion, we have shown for the first time that (i) TRAIL-Rs are expressed on human adult glial cells and (ii) TRAIL can induce selective ahOL apoptotic cell death which is dependent on protein synthesis inhibition and ligation of TRAIL-R1. The selectivity of TRAIL susceptibility of ahOL seems to be relevant to deficient expression of the decoy receptor TRAIL-R3. These results may be relevant to the CNS inflammatory/demyelinating conditions where the demise of oligodendrocytes occurs and may contribute to the development of new molecules that interfere with the immunopathogenesis of these diseases.
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Acknowledgements |
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We wish to thank Dr Tony Troutt, Immunex, Seattle WA, USA for his generous gift of TRAIL-LZ and antibodies against TRAIL and TRAIL-Rs, and Dr Marek Kubin, Immunex, for providing us with additional information on TRAIL antibodies. This work was supported by KBN grants 4PO5A 006.14, 4PO5A 083.18 and MU grant 502-11-368.
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