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The Journal of Immunology, 1999, 162: 2326-2333.
Copyright © 1999 by The American Association of Immunologists

CD95 (Fas/Apo-1) as a Receptor Governing Astrocyte Apoptotic or Inflammatory Responses: A Key Role in Brain Inflammation?1

Philippe Saas2,*, José Boucraut{ddagger}, Anne-Lise Quiquerez*, Valérie Schnuriger*, Gaelle Perrin*, Sophie Desplat-Jego{ddagger}, Dominique Bernard{ddagger}, Paul R. Walker*,{dagger} and Pierre-Yves Dietrich3,*

* Laboratory of Tumor Immunology, Division of Oncology, and {dagger} Department of Neurosurgery, University Hospital, Geneva, Switzerland; and {ddagger} Laboratory of Immunopathology, Faculty of Medicine, Marseille, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Astrocytes are a major cellular component of the brain that are capable of intense proliferation and metabolic activity during diverse inflammatory brain diseases (such as multiple sclerosis, Alzheimer’s dementia, tumor, HIV encephalitis, or prion disease). In this biological process, called reactive gliosis, astrocyte apoptosis is frequently observed and could be an important mechanism of regulation. However, the factors responsible for apoptosis in human astrocytes are poorly defined. Here, we report that short term cultured astrocytes derived from different brain regions express significant levels of CD95 at their surface. Only late passage astrocytes are sensitive to CD95 ligation using either CD95 mAb or recombinant CD95 ligand. Blocking experiments using caspase inhibitors with different specificities (DEVD-CHO, z-VAD-fmk, and YVAD-cmk), an enzymatic activity assay, and immunoblotting show that CPP32/caspase-3 play a prominent role in CD95-induced astrocyte death. In contrast, early passage astrocytes are totally resistant to death, but a significant increase in astrocytic IL-8 secretion (p < 0.001, by Wilcoxon’s test for paired samples) is observed after CD95 triggering. Production of IL-8 contributes to the resistance of astrocytes to CD95 ligation. Furthermore, in the presence of IFN-{gamma}, resistant astrocytes became sensitive to CD95-mediated death. These data suggest that microenvironmental factors can influence the consequences of CD95 ligation on astrocytes. Therefore, we propose that CD95 expressed by human astrocytes plays a pivotal role in the regulation of astrocyte life and death and may be a key factor in inflammatory processes in the brain, such as reactive gliosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Astrocytes belong to the family of glial cells and were long considered to have a passive role in maintaining brain structure. However, recent data indicate the pivotal role of these cells in physiological and pathological processes in the central nervous system (CNS)4, 1 , in which the regulation of astrocyte survival is often critical. During development, massive cell death is needed to achieve mature functional and anatomical regions. The cell types implicated were not always defined 2, 3 , but now it is clear that both neurons and astrocytes die 2, 4 . During inflammation, there is also significant astrocyte death that occurs after a period of proliferation of nontumor-reactive astrocytes (reactive gliosis) 5 . Indeed, any injury (infection, autoimmunity, tumor, or stroke) can induce such gliosis, the intensity and duration of which may be limited by astrocyte apoptosis, as reported in HIV encephalitis 6, 7 , Alzheimer’s disease 8, 9 , and multiple sclerosis (MS) 10 .

Apoptosis (active programmed cell death) can be induced by diverse stimuli. A key molecule is CD95 (Fas/Apo-1), a transmembrane protein belonging to the TNF receptor family that transduces apoptotic signals after ligation by agonistic Abs or by its natural ligand (CD95L/FasL), a member of the TNF family 11 . Here, we examine whether CD95 may contribute to human astrocyte apoptosis by using cultured astrocytes derived from human embryos. In this culture system, a high level of purity is obtained after three passages, allowing the study of functional parameters on glial fibrillary acidic protein (GFAP) positive astrocytes 12, 13, 14 . We show that human astrocytes express CD95 that can transduce an apoptotic signal or an activation signal, leading to proinflammatory cytokine release. The particular outcome is governed by the presence of IFN-{gamma}, indicating that in vivo, microenvironmental factors and cellular interactions can alter the course and duration of inflammatory brain diseases by effects on the CD95 pathway of astrocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Abs

mAbs against human GFAP (6F2, mouse IgG1) and CD3 (4B5, mouse IgG1, negative control for GFAP staining), anti-GFAP polyclonal antiserum, FITC-conjugated anti-human Bcl-2 mAb (124, mouse IgG1), FITC-conjugated mouse IgG1 (negative control mAb for Bcl-2 detection), and an FITC-conjugated F(ab')2 of goat anti-mouse Ig were obtained from Dako (Zug, Switzerland). The lytic anti-human CD95 mAb (CH11, mouse IgM), phycoerythrin (PE)-conjugated anti-human CD95 mAb (UB2, mouse IgG1), and Cy3-conjugated F(ab')2 of goat anti-rabbit Ig were obtained from Immunotech (Marseille, France). Mouse IgM anti-human CD57 mAb HNK-1 (ATCC TIB200) and PE-conjugated anti-human CD8 mAb (mouse IgG1; Serotec, Oxford, U.K.) were used as isotype-matched negative control mAb for CD95 ligation and CD95 staining, respectively. Recombinant CD95L was obtained from supernatant of CD95L-transfected Neuro-2a cells 15 . Supernatant from mock-transfected Neuro-2a cells was used as control. These cells were provided by A. Fontana (Zurich, Switzerland). Caspase inhibitors z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) and YVAD-cmk (acetyl-Tyr-Val-Ala-Asp-chloromethylketone) were obtained from Bachem Biosciences (Budendorf, Switzerland). Human rIFN-{gamma} and neutralizing anti-human IL-8 mAb (mouse IgG1) were obtained from R & D Systems (Abingdon, U.K.). Anti-human CD3 mAb (mouse IgG, OKT3), provided by Drs. P. Tiberghien and E. Robinet (Besançon, France), was used as an isotype control for anti-IL8 mAb. Human rTNF-{alpha} was provided by C. Ruegg (Lausanne, Switzerland).

Established cell lines

Astrocytoma cell lines LN18, LN215, and LN229 were provided by N. de Tribolet and E. G. Van Meir (Lausanne, Switzerland). The human cell line Jurkat (ATCC TIB152) was obtained from the American Type Culture Collection (Manassas, VA). The human pancreatic cancer cell line Colo357 cDNA was provided by H. Kalthoff and H. Ungefroren (Kiel, Germany).

Human astrocyte culture

Primary human astrocyte cultures were prepared as previously described 12, 13, 14 from fetal abortions (7–9 wk). Tissue was procured in accordance with institutional regulations. Independent cultures were established from each brain specimen. The myelencephalon and mesencephalon, telencephalon, or spinal cord were isolated under sterile conditions. After removal of meninges, the tissue was dissociated by trypsinization and mechanical disruption. The cells were seeded on poly-L-lysine (10 µg/ml; Sigma, St. Louis, MO)-coated flasks at a density of 2 x 103 cells/cm2 and grown at 37°C in a humidified atmosphere with 5% CO2 in MEM D-valine (Life Technologies, Grand Island, NY) supplemented with 5% FCS, 2 mM L-glutamine (Life Technologies), 20 ng/ml EGF (Sigma), 50 µg/ml apotransferrin (Sigma), 5 µg/ml insulin (Sigma), 100 IU/ml penicillin (Life Technologies), and 100 µg/ml streptomycin (Life Technologies). After 1 mo of culture, a confluent layer of GFAP-positive cells was obtained. Cells were then passaged using 0.25% trypsin-EDTA (Life Technologies) every 3 wk and were replated at a 1/3 dilution. The expression of GFAP and the absence of mycoplasma (ELISA detection kit, Boehringer Mannheim, Rotkreuz, Switzerland) were confirmed for all cultures.

Flow cytometry

Analysis of astrocytes was performed on a FACScan (Becton Dickinson, Mountain View, CA) using LYSYS II software. Cells were detached from culture vessels using brief exposure to trypsin-EDTA and were stained with the appropriate mAb or isotype-matched control mAb. Dead cells were excluded by forward and side scatter or propidium iodide (Sigma) gating. For intracytoplasmic staining, cells were fixed in 0.25% paraformaldehyde (Fluka, Buchs, Switzerland) in PBS and permeabilized in 70% cold ethanol before the staining procedure.

RT-PCR analysis

Total RNAs isolated from astrocytes using TRIzol reagent (Life Technologies) were converted to cDNA by standard methods using reverse transcriptase (Life Technologies) and an oligo(dT) primer (Life Technologies). These cDNA were amplified in nonsaturating PCR conditions using specific primers. Primer sequences (sense primers are indicated first) are as follows: ß-actin, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' (positions 541–569) and 5'-CTAGAAGCATTTGCGGTGGACGACGATGGAGGG-3' (positions 1172–1201); CD95, 5'-ATGCTGGGCATCTGCACCCT-3' and 5'-TCTAGACCAAGCTTTGGATTTC-3' 16 ; Fas-associated phosphatase-1 (FAP-1), 5'-GAATACGAGTGTCAGACATGG-3' and 5'-AGGTCTGCAGAGAAGCAAGAATAC-3' 17, 18 ; and Bcl-x, 5'-GGAGTCAGTTTAGTGATGTG-3' (positions 205–224) and 5'-CTGCATTGTTCCCATAGAGT-3' (positions 713–732). After amplification, PCR products were separated by electrophoresis on agarose gel containing ethidium bromide and visualized by UV light illumination.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay

Astrocytes obtained from mesencephalon/myelencephalon were seeded into poly-L-lysine-coated wells of 24-well plates at 4 x 104/well and incubated for 16 h to obtain cell adherence. Then, cells were incubated with lytic anti-CD95 mAb CH11 or control IgM, TNF-{alpha}, C2 ceramide (Sigma), t-butyl hydroperoxide (Sigma), or neutralizing anti-IL-8 mAb (at the indicated concentrations) for 24 h. Cell viability was determined by the MTT assay, which determines mitochondrial activity in living cells 19 . Results are expressed as the percentage of dead cells = 100 - [(anti-CD95 mAb-treated astrocyte OD550/control IgM-treated astrocyte OD550) x 100].

Detection of apoptosis by cell cycle analysis

Apoptotic cells were detected by propidium iodide staining and flow cytometry as previously reported 20 . Briefly, astrocytes were fixed in 1% paraformaldehyde (Fluka) in PBS and permeabilized in 70% cold ethanol before the staining procedure. Then, cells were incubated with propidium iodide (50 µg/ml) and RNase A (50 µg/ml; Boehringer Mannheim, Indianapolis, IN) and were analyzed by flow cytometry.

Caspase activity assay

Cells were treated for 6 h (defined in Fig. 3Go) and lysed in ice-cold hypotonic solution consisting of 20 mM Tris-HCl (pH 7.2), 1 mM EDTA (Sigma), and 10 µg/ml trypsin inhibitor (Sigma). Then, homogenates were clarified by centrifugation at 13,000 rpm at 4°C, and supernatants were collected and diluted in 50 mM Tris-HCl (pH 7.5), 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic, and 10 mM DTT (Boehringer Mannheim). Enzymatic reactions were conducted in triplicate in a total volume of 200 µl containing lysate from 105 cells and 100 µM DEVD-p-nitroaniline (succinyl-Asp-Glu-Val-Asp-pNA) or 100 µM YVAD-pNA (acetyl-Tyr-Val-Ala-Asp-pNA) (Alexis Corp., Laufelfingen, Switzerland) in the presence or the absence of the specific inhibitor DEVD-CHO (succinyl-Asp-Glu-Val-Asp-aldehyde) or YVAD-CHO (acetyl-Tyr-Val-Ala-Asp-aldehyde; Alexis Corp.). The release of pNA was determined by measuring the OD at 405 nm.



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  		FIGURE 3. CD95 ligation induces astrocyte death via CPP32/caspase-3 subfamily activation. A, Astrocyte death after CD95 ligation requires inhibition of protein synthesis and is prevented by z-VAD-fmk and DEVD-CHO. Early passage astrocytes were pretreated with CHx (10 µg/ml), then CH11 mAb or control mAb (1 µg/ml) was added, and cell viability was determined using the MTT assay. Pretreatment with caspase peptide inhibitors z-VAD (z-VAD-fmk, 50 µM) or DEVD (DEVD-CHO, 100 µM), but not with YVAD (YVAD-cmk, 200 µM), protects astrocytes from CD95 killing. Data from a representative experiment (of a total of four) are shown. The percentage of dead cells was calculated as described in Materials and Methods. Error bars represent the SD. B, CPP32/caspase-3-like activity is detected only in cell lysates of CD95-sensitive astrocytes (i.e., those treated with CHx and CH11 mAb). Enzymatic activity in cell lysates was determined using CPP32/caspase-3 subfamily-specific substrate DEVD-pNA as described in Materials and Methods. Astrocytes were stimulated under different conditions: CHx plus control mAb (open circles), CH11 mAb alone (open squares), and CHx plus CH11 mAb (filled circles). Preincubation of CD95-sensitive astrocytes with the CPP32/caspase-3 subfamily inhibitor DEVD-CHO (50 µM; filled triangles) prevented DEVD-pNA cleavage, whereas the ICE/caspase-1 subfamily inhibitor YVAD-CHO (200 µM; open triangles) remained ineffective. CHx and mAbs were used at final concentrations of 10 and 1 µg/ml, respectively. Data from a representative experiment (of three) are shown. The SD are <0.009 OD. C, CPP32/caspase-3 is processed in CD95-sensitive astrocytes into p17- and p20-active subunits. Astrocytes were treated for 12 h under different conditions: anti-CD95 mAb (1 µg/ml) alone, CHx (10 µg/ml) plus IgM (1 µg/ml; noted CHx on the figure), anti-CD95 mAb (1 µg/ml) plus CHx (10 µg/ml), or IFN-{gamma} (100 IU/ml, 24 h) plus anti-CD95 mAb. CPP32/caspase-3 cleavage in the CD95-sensitive cell line Jurkat is shown as a positive control.

 
CPP32/caspase-3 immunoblot

One million astrocytes were lysed in ice-cold hypotonic lysis buffer as previously described 21 . Proteins were separated by SDS-PAGE under reducing conditions, then transferred to nitrocellulose. CPP32/Caspase-3 was detected with a specific mAb (mouse IgG2a, Transduction Laboratories, Exeter, U.K.) followed by peroxidase-conjugated rabbit anti-mouse mAb (Dianova, Hamburg, Germany) and visualized by chemiluminescence reaction using the ECL system (Amersham, Aylesbury, U.K.).

IL-8 production

Astrocytes obtained from mesencephalon/myelencephalon were stimulated with CH11 mAb or control IgM for 24 h. Then, supernatant was collected, and IL-8 production was measured using an ELISA kit (R & D Systems) according to the manufacturer’s instructions. Data were analyzed using Wilcoxon’s test for paired samples 22 .


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human cultured astrocytes express CD95

Cells derived from mesencephalon and myelencephalon showed typical astrocytic morphology, with >than 90% of the cells expressing the astrocyte-specific marker GFAP (Fig. 1Go, A and B). CD95 expression was first analyzed by RT-PCR. A PCR product of 1008 bp corresponding to the full-length CD95 cDNA and a smaller transcript of 945 bp corresponding to CD95 splicing variant mRNA (called Fas{Delta}TM in 23 were clearly identified in astrocytes at all passages tested (Fig. 1GoC). Since the expression pattern of cell surface glycoproteins may be heterogeneous in astrocytes isolated from different brain regions 24 , CD95 cell surface expression was analyzed in astrocytes derived from the mesencephalon/myelencephalon, the telencephalon, and the spinal cord. There was a conservation of CD95 expression not only in all astrocyte cultures tested (Fig. 1GoD), but also throughout the culture period (passages 3–12) (data not shown).



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  		FIGURE 1. GFAP-positive human astrocytes express CD95. A, GFAP expression by astrocytic cultures (after four passages in vitro) from human embryonic mesencephalon/myelencephalon. For intracellular detection of GFAP, cells were stained with anti-GFAP mAb 6F2 (filled curves) or with control mAb 4B5 (open curves) followed by incubation with a secondary FITC-conjugated anti-Ig and were analyzed by flow cytometry. B, Cultures from embryonic mesencephalon/myelencephalon (fourth passage) show typical astrocytic morphology with GFAP expression. Cultures grown on glass coverslips were fixed and stained with anti-GFAP polyclonal antiserum, revealed with Cy3-conjugated mAb, and analyzed by fluorescence microscopy (final magnification, x175). C, Expression of CD95 mRNA transcripts corresponding to the full-length CD95 cDNA (1008 bp) and the splicing variant mRNA (945 bp). RT-PCR was performed on total RNA from astrocytes at different passage numbers (P) in culture. Positive controls were PHA-activated (24 h) PBMC (PBMC*) and astrocytoma cell lines LN215 and LN229. D, Cell surface CD95 expression on human cultured astrocytes obtained from different brain regions. Cells stained with PE-conjugated anti-CD95 mAb UB2 (filled curves) or PE-conjugated control mAb (open curves) were analyzed by flow cytometry. Experiments were repeated at least three times with reproducible results.

 
CD95 ligation may induce astrocyte death

To test the outcome of CD95 ligation, the percentage of dead cells among early passage astrocytes (<7 passages) incubated with lytic anti-CD95 mAb or recombinant CD95L was assessed in an MTT cell viability assay. These cells were totally resistant, even with increasing mAb concentrations (Fig. 2Go) or prolonged time of exposure (until 72 h). This resistance is an active process, since astrocytes pretreated with cycloheximide (CHx; a protein synthesis inhibitor that did not affect CD95 expression or astrocyte viability; data not shown) were highly sensitive to CD95 mAb-induced death (Fig. 3GoA). After 8–10 passages in culture (late passage), astrocytes became sensitive to anti-CD95 mAb-mediated cell death (Fig. 2Go). Similar results were obtained with recombinant CD95L rather than CH11 mAb (data not shown). However, these cells remained resistant to TNF-{alpha} or oxidative stress (data not shown).



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  		FIGURE 2. Early passage astrocytes are resistant to anti-CD95 mAb-mediated death, whereas late passage are sensitive to lytic anti-CD95 mAb. Early passage astrocytes (open bars) and late passage astrocytes (solid bars) were incubated with different concentrations of CH11 mAb or control IgM, and cell viability was assessed using the MTT assay. Data from a representative experiment (of a total of four) are shown. The percentage of dead cells was calculated as described in Materials and Methods. Error bars represent the SD.

 
CD95-mediated apoptosis requires CPP32/caspase-3 activation

The same experiments using CD95-sensitive astrocytes or CHx-treated astrocytes were repeated in the presence of caspase inhibitors with different specificities. While YVAD-cmk preferentially inhibits protease of the ICE/caspase-1 subfamily, DEVD-CHO is a selective inhibitor of the CPP32/caspase-3 subfamily, and z-VAD-fmk is an inhibitor with broader activity 11, 25 . CD95-induced death of astrocytes was totally suppressed in the presence of z-VAD-fmk or DEVD-CHO, but persisted with YVAD-cmk (Fig. 3GoA). This suggests a preferential involvement of the CPP32/caspase-3 subfamily in CD95 astrocyte death. To extend these data, we measured CPP32/caspase-3-like enzymatic activity in the cell lysates of early passage astrocytes treated under different conditions (Fig. 3BGo). There was no significant cleavage of the colorimetric substrate DEVD-pNA by lysates of either unstimulated astrocytes (data not shown) or cells incubated with CD95 mAb or CHx plus control IgM. In contrast, significant cleavage was observed in lysates from dying astrocytes (i.e., those cultured in the presence of CD95 mAb and CHx), an effect totally abrogated by DEVD-CHO but not by YVAD-CHO. The direct involvement of CPP32/caspase-3 in the CD95-mediated death of astrocytes was confirmed by Western blot analysis (Fig. 3GoC). A colorimetric assay using ICE/caspase-1 subfamily-specific substrate YVAD-pNA was also performed, but no activation of ICE/caspase-1 was detected in lysates of cells pretreated with CHx and CD95 mAb (data not shown).

CPP32/caspase-3 is responsible for proteolysis of substrates involved in DNA repair. This cleavage contributes to nuclear alterations associated with apoptosis 25 . Thus, apoptosis was analyzed using propidium iodide staining and flow cytometry (Fig. 4Go, A–C). Significant DNA fragmentation was found in astrocytes treated for 14 h with CD95 mAb CH11 or rCD95L in the presence of CHx. Subdiploid nuclei (corresponding to apoptotic cells) were reduced when astrocytes were pretreated by the CPP32/caspase-3 family-specific inhibitor DEVD-CHO (Fig. 4GoC). Thus, DNA fragmentation in sensitive astrocytes is induced after CD95 ligation and is subsequent to CPP32/caspase-3 activation.



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  		FIGURE 4. CD95 induces apoptosis in CHx- or IFN-{gamma}-treated astrocytes. Cells were treated with 1 µg/ml anti-CD95 mAb CH11 (A), CH11 mAb plus 10 µg/ml CHx (B), 50 µM DEVD-CHO plus CH11 mAb and CHx (C), or 100 IU/ml IFN-{gamma} plus CH11 (D). After 12 h, cells were fixed and permeabilized. DNA was stained with propidium iodide and analyzed by flow cytometry. The percentage of apoptotic nuclei is indicated on each graph. Apoptotic nuclei are represented by the subG1 peak. Data from a representative experiment (of three) are shown.

 
Potential mechanisms implicated in CD95 resistance

Possible mechanisms that may contribute to the resistance of early passage astrocytes to CD95-mediated death were then investigated. A first hypothesis was the secretion of a functional recombinant CD95 molecule (Fas{Delta}TM) that could neutralize CD95 agonists 16, 23 , since the 945-bp transcript that codes for this form was clearly identified in all cultured astrocytes (Fig. 1GoC). Supernatants collected from early or late passage astrocytes did not protect CD95-sensitive Jurkat cells from death (data not shown), making this hypothesis unlikely. Another possibility was a differential expression of Bcl-2 family members that have been implicated in the control of cell death in the astrocyte lineage 26, 27 . In the human astrocyte cultures tested here, Bcl-2 was expressed, but there was no correlation between expression level and CD95 resistance (Fig. 5GoA). The mRNA expression of both Bcl-xL and Bcl-xS isoforms was similar in all astrocytes tested (Fig. 5GoB). A particularly interesting candidate to further test in CD95-resistant cells was FAP-1, since this molecule directly and specifically interacts with the negative cytoplasmic domain of CD95 17 . FAP-1 transcript was highly expressed in CD95-resistant (early passage) astrocytes, whereas only a faint RT-PCR signal was obtained in CD95-sensitive (late passage) astrocytes (Fig. 5GoC). The present data suggest that FAP-1 mRNA expression is correlated with astrocyte resistance to CD95-induced death.



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  		FIGURE 5. Potential mechanisms involved in astrocyte CD95 resistance. A, Similar levels of Bcl-2 are expressed by early passage (CD95-resistant, CD95R) or late passage (CD95-sensitive, CD95S) astrocytes. For intracellular detection of Bcl-2, cells were stained with FITC-conjugated anti-Bcl-2 mAb or FITC-conjugated control mAb and analyzed by flow cytometry. Previously characterized human astrocytoma cell lines (LN18 and LN215) (41) were used as controls. B, Early passage (P4 to P7) and late passage (P8 and P10) astrocytes display similar Bcl-xL mRNA expression (508-bp RT-PCR product). A faint band at 319 bp corresponding to the Bcl-xS isoform was detected in all samples. Human astrocytoma cell lines (LN215 and LN229), PHA-activated PBMC (PBMC*), and brain biopsy (Ge60, obtained from epilepsy surgery) were used as positive controls; the Jurkat cell line served as the negative control. C, FAP-1 mRNA expression is correlated with CD95 resistance. RT-PCR under nonsaturating conditions was performed on total RNA from CD95-resistant (FasR) and CD95-sensitive (FasS) astrocytes. Results from two independent experiments are shown (noted FasR1/FasS1 and FasR2/FasS2). Human astrocytoma (LN215 and LN229) and pancreatic carcinoma (Colo 357) cell lines were used as positive controls, epileptic brain biopsy (Ge60) was weakly positive, and Jurkat cells served as the negative control. Amplification of a 660-bp ß-actin fragment is also shown.

 
CD95 triggering on astrocytes augments IL-8 secretion

In addition to apoptosis, CD95 can deliver activation signals 28, 29, 30 . Cultured astrocytes refractory to CD95-induced death were therefore attractive candidates to test for other consequences of CD95 ligation. Basal levels of IL-8 were detected in the supernatants of unstimulated astrocytes, as previously described 13 . CD95 ligation using CH11 mAb significantly (p < 0.001) enhanced IL-8 secretion (Fig. 6Go), with similar results obtained using recombinant CD95L (data not shown). Interestingly, an increase in IL-8 secretion was detected only in early passage astrocytes (i.e., CD95 resistant), and astrocytes pretreated with neutralizing anti-IL-8 mAb became sensitive to CD95-mediated death (Fig. 7Go). This strongly suggests that IL-8 contributes to astrocyte resistance to CD95-induced apoptosis. Overall, these data demonstrate that the same receptor (CD95) can mediate different cellular functions (i.e., apoptosis or cytokine secretion) depending upon the status of the astrocytes tested.



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  		FIGURE 6. CD95 triggering induces IL-8 secretion. Early passage astrocytes from embryonic mesencephalon/myelencephalon (fourth passage) were stimulated with CH11 mAb or control mAb (1 µg/ml) for 24 h. IL-8 present in supernatant was measured using an ELISA kit. Twelve independent experiments from three separate embryos were performed. p < 0.001, by Wilcoxon’s test for paired sample.

 


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  		FIGURE 7. IL-8 is involved in the resistance of astrocyte to CD95 ligation. Astrocytes (fourth passage) were incubated for 24 h with CH11 mAb or control IgM (1 µg/ml) in the presence of anti-IL-8 mAb or control IgG1 (5 µg/ml). Cell viability was assessed by the MTT assay. Data from three independent experiments are shown. The percentage of dead cells was calculated as described in Materials and Methods. Error bars represent the SD.

 
IFN-{gamma} as a switch factor that influences the astrocyte response to CD95

To determine whether the outcome of CD95 ligation may be influenced by microenvironmental factors, we examined the effects of IFN-{gamma}. This cytokine was chosen because it can potentiate CD95-mediated apoptosis in certain cell types and increase CD95 expression on the cell surface 11 . IFN-{gamma} alone did not modify the viability of astrocytes, but preincubation (100 IU/ml, 24 h) rendered them sensitive to CD95-mediated apoptosis (Figs. 4GoD and 8A). This effect was abrogated by incubation with z-VAD-fmk (Fig. 8GoA). In addition, CPP32/caspase-3 cleavage was detected by immunoblot analysis after IFN-{gamma} pretreatment and CD95 ligation (Fig. 3GoC), demonstrating that this caspase is required for CD95-induced death of IFN-{gamma}-treated cells. Whether the sensitizing effect of IFN-{gamma} is restricted to the CD95 pathway or is applicable to other apoptotic stimuli to which astrocytes in culture show resistance 31, 32, 33 was another question to address. After IFN-{gamma} treatment, astrocytes remained totally insensitive to TNF-{alpha} or oxidative stress-induced death, and only a small augmentation of ceramide-mediated death was noted (Fig. 8GoB). Why IFN-{gamma} preferentially operates on the CD95 pathway is not known. An increase in cell surface CD95 expression was observed after IFN-{gamma} treatment (Fig. 8GoC). However, it is unlikely that this is the sole mechanism involved, since IFN-{gamma} also induces a slight augmentation of type 1 TNF-{alpha} receptor (TNFR1/CD120a) expression (data not shown) without inducing sensitivity to TNF-{alpha}-mediated death.



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  		FIGURE 8. IFN-{gamma} reverses astrocyte resistance to CD95-mediated cell death. A, IFN-{gamma} pretreatment induces astrocyte death after CD95 triggering, and caspase activity is required for CD95-induced death of IFN-{gamma}-treated cells. After 24-h incubation with 100 IU/ml of human IFN-{gamma}, CH11 mAb or control mAb (at 1 µg/ml) was added, and cell viability was assessed by the MTT assay. Caspase inhibitor z-VAD (z-VAD-fmk, 50 µM) was used as previously described in Fig. 3GoA. B, The sensitizing effect of IFN-{gamma} is restricted to CD95. After incubation with IFN-{gamma} (as described above), astrocytes were exposed to cell-permeable ceramide (C2 ceramide, 50 µM), TNF-{alpha} (10 ng/ml), or oxidative stress (Ox. stress) using t-butyl hydroperoxide (50 µM), and cell viability was determined. Results were compared with those obtained with anti-CD95 mAb CH11. Data from a representative experiment (of three) are shown in A and B. Cell death was calculated as described in Materials and Methods. Error bars represent the SD. C, IFN-{gamma} increases astrocyte CD95 cell surface expression. Untreated and IFN-{gamma}-treated (100 IU/ml) passage 4 astrocytes were stained either with PE-conjugated anti-CD95 mAb UB2 (filled curves) or with PE-conjugated control mAb (open curves) and were analyzed by flow cytometry.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent in vitro data suggest that astrocytes may play a significant role in the immune responses taking place in the CNS 12, 13, 14, 34, 35 . The use of astrocytes in culture is a valuable approach to study the function of cell surface molecules on a purified population and to extend descriptive ex vivo analysis. For example, astrocytes have been shown to produce various cytokines after appropriate stimulation mimicking in vivo conditions 13, 14 . Furthermore, these cells can respond to cytokines such as TNF-{alpha} or IFN-{gamma} with enhanced expression of adhesion molecules 12, 36 and acquisition of MHC class II expression, suggesting that they may potentially function as APCs 34 . Here, we report that human cultured astrocytes derived from different areas of the brain express significant levels of CD95. This receptor can mediate either death via CPP32/caspase-3 cleavage or proinflammatory cytokine release depending upon the astrocyte activation state. The final outcome of the astrocyte response to CD95 ligation can be influenced by microenvironmental factors, since IFN-{gamma} rendered resistant astrocytes susceptible to CD95-mediated death. Our results are consistent with CD95 expression by astrocytes being important in the regulation of inflammatory responses associated with many brain diseases.

Until now, most of our understanding about the role of the CD95 system and its signaling pathway has been obtained with cells of the immune system. However, it is clear that information concerning one cell type cannot be directly applied to other cells. Indeed, intracellular events conferring resistance to CD95 triggering are cell specific and can be modified for example after differentiation 37 . In the same way, the second messengers responsible for the execution phase of apoptosis may also depend upon cell type 38, 39, 40, 41 , but no information was available to date for astrocytes. Here we show that astrocytes resistant to death mediated by CD95 ligation express abundant FAP-1, whereas FAP-1 was barely detectable in sensitive astrocytes. Such a correlation between FAP-1 mRNA expression and resistance to CD95-induced apoptosis was also recently reported in Th1 and Th2 lymphocytes 37 and in some CD95-positive tumor cell lines 17, 18 . The present data suggest that this mechanism also occurs in astrocytes, although FAP-1 may not be the sole molecule involved, since FAP-1 mRNA expression was not significantly modified after IFN-{gamma} treatment (data not shown). Furthermore, we clearly show the involvement of CPP32/caspase-3 in CD95-mediated astrocyte death. This observation is particularly relevant, since CPP32/caspase-3 deficient mice die at 1–3 wk of age, with accumulation of neurons and GFAP-positive cells due to defective cell death 42 . An increase in brain cell number has not been reported to date for lpr or gld mice (i.e., mice with a nonfunctional CD95/CD95L system), suggesting that multiple mechanisms converging through CPP32/caspase-3 exist in the CNS to control astrocyte death during development. However, MRL/lpr mice present behavioral dysfunctions 43, 44 , suggesting a role for the CD95 signaling pathway during CNS development.

In certain cell types, CD95 ligation delivers not only a death signal but also activation signals 28, 29, 30 , although no data exist for resident cells of the brain. One potentially important function of astrocytes is the release of cytokines and chemokines, among which IL-8 is of particular interest. Indeed, IL-8 may promote inflammation 45 and angiogenesis 46 in the CNS and may also support neuronal survival 47 following brain injury. However, the activation signals augmenting IL-8 release by astrocytes are not fully defined. Previous studies have reported that IL-1ß and TNF-{alpha}, but not LPS, induced IL-8 production by human cultured astrocytes 13, 48 . Here, we demonstrate that CD95 ligation significantly enhanced IL-8 secretion by astrocytes. Although the production of IL-8 through CD95 stimulation has been previously reported for other cell types 29, 49 , implication of this cytokine in resistance to CD95-induced apoptosis was unknown. In this study, IL-8 was identified as an astrocyte survival factor. Further investigations currently in progress should give insights into the second messengers of the CD95 pathway that are affected by IL-8. In the brain, the IL-8 detected during viral and bacterial infections or in the presence of malignancies 45 may thus be the product of CD95L-stimulated reactive astrocytes. We propose that the regulation of IL-8 secretion via the CD95 pathway participates in the pathogenesis of many brain diseases.

The dramatic consequences induced by CD95 ligation on astrocytes need to be considered in the context of the biological source of CD95L in normal and pathological conditions. Trafficking leukocytes include CD95L-expressing cells that could mediate important interactions 50 , particularly in the context of inflammation and tumors. Furthermore, CNS resident cells (neurons or glial cells) may potentially express CD95L, although contradictory results have been reported 10, 51, 52, 53, 54 . Nevertheless, CD95 and CD95L have recently been implicated in the pathogenesis of experimental autoimmune encephalomyelitis 55, 56, 57 . In addition to the cellular origin of CD95L, it is important to consider factors present in the astrocyte microenvironment that may influence the final consequences of CD95 ligation. IFN-{gamma} was an interesting candidate to test, since this cytokine is released from activated T lymphocytes during CNS autoimmune and infectious diseases 58, 59, 60, 61, 62 . Moreover, recent data suggest that neurons could be an additional source of IFN-{gamma} 63 . This cytokine is also implicated in cellular differentiation during brain development 64 . We propose that IFN-{gamma} plays a pivotal role in the functions (inflammation and apoptosis) mediated by CD95, since astrocytes cultured in the presence of IFN-{gamma} for 24 h are switched to respond to CD95 ligation by apoptosis. This shows that microenvironmental factors produced by neurons and infiltrating inflammatory cells can influence the central role of CD95 in astrocyte survival.

Overall, the present findings implicate that CD95 is an important component in the complex control of CNS inflammation. The normal brain is protected from overwhelming and deleterious immune responses by several passive (e.g., physical isolation by blood-brain barrier) and active (e.g., release of immunosuppressive factors) mechanisms. However, after brain injury certain immune responses do proceed in the CNS, but they must be tightly regulated. We propose that CD95 expressed by astrocytes could perform this role during inflammatory responses that are associated with reactive gliosis. Indeed, cultured astrocytes share phenotypic and functional characteristics with reactive astrocytes seen in various diseases (Alzheimer’s disease, MS, stroke, HIV infection, and tumors). Both reactive and cultured astrocytes express high levels of GFAP and show a high proliferation index at initial stages 5, 12, 65 . Moreover, human (reported here and in 54 or murine 55 astrocytes in culture as well as reactive astrocytes 66 express CD95, whereas resting astrocytes, either by immunochemistry 66, 67 or analyzed ex vivo (data not shown) are negative. At early stages of culture, ligation of CD95 on astrocytes leads to enhanced secretion of IL-8. Such a mechanism could be responsible for perpetuating inflammatory responses in vivo. The final outcome for cultured astrocytes is death by spontaneous apoptosis (data not shown), and indeed, reactive astrocyte apoptosis has been reported in many brain diseases 6, 7, 8, 9, 10 , colocalizing with CD95 staining in MS lesions 10 . Inflammatory cytokines such as IFN-{gamma} may influence the response of reactive astrocytes to CD95L, since IFN-{gamma} sensitizes astrocytes to CD95 triggering. Consistent with these findings, it has recently been reported that intracerebral injection of IFN-{gamma} limits reactive astrocyte expansion following physical brain injury 68 . Unraveling the complexities of the CD95 pathway in astrocytes should shed light on the phenomenon of reactive gliosis and clarify issues concerning the control of astrocyte number in development.


   Acknowledgments
 
We thank M. Barnet, Y. Chicheportiche, I. Desbaillets, C. Favre, A. Fontana, H. Kalthoff, V. Kindler, P. Paul, E. Robinet, P. Tiberghien, H. Ungefroren, and E. G. Van Meir for reagents. We acknowledge B. Mermillod for statistical analysis and N. de Tribolet, T. Landis, D. Bertrand, and A.-P. Sappino for helpful discussions.


   Footnotes
 
1 This work was supported by grants from the Swiss National Science Foundation (31-50585.97 to P.-Y.D.), the Helmut Horton Stiftung (to P.S), the Ligue Genevoise contre le Cancer (to P.R.W.), the Ligue Suisse contre le Cancer, the Sir Jules Thorn Charitable Overseas Trust Reg. Schaan, the CIBA-Geigy-Jubiläums Stiftung, the Ligue Française contre le Cancer, and the Fondation pour la lutte contre le Cancer (to P.-Y.D.). Back

2 Current address: Etablissement de Transfusion Sanguine de Franche-Comté, Laboratoire de Thérapeutique Immuno-Moléculaire, 1 bld A. Fleming, F-25020 Besançon, France. Back

3 Address correspondence and reprint requests to Dr. Pierre-Yves Dietrich, Laboratory of Tumor Immunology, Division of Oncology, University Hospital, rue Micheli-du-Crest 24, CH-1211 Geneva 14, Switzerland. E-mail address: Back

4 Abbreviations used in this paper: CNS, central nervous system; MS, multiple sclerosis; GFAP, glial fibrillary acidic protein; PE, phycoerythrin; CD95L, CD95 (Fas/Apo-1) ligand; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; YVAD-cmk, acetyl-Tyr-Val-Ala-Asp-chloromethylketone; FAP-1, Fas-associated phosphatase-1; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; DEVD-pNA, succinyl-Asp-Glu-Val-Asp-p-nitroaniline; YVAD-pNA, acetyl-Tyr-Val-Ala-Asp-pNA; DEVD-CHO, succinyl-Asp-Glu-Val-Asp-aldehyde; YVAD-CHO, acetyl-Tyr-Val-Ala-Asp-aldehyde; CHx, cycloheximide; ICE, IL-1ß-converting enzyme. Back

Received for publication June 18, 1998. Accepted for publication November 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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