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[Cancer Research 61, 3084-3091, April 1, 2001]
© 2001 American Association for Cancer Research

Immunology

Fas-induced Expression of Chemokines in Human Glioma Cells

Involvement of Extracellular Signal-regulated Kinase 1/2 and p38 Mitogen-activated Protein Kinase1

Chulhee Choi, Xiang Xu, Jae-Wook Oh, Sung Joong Lee, G. Yancey Gillespie, Heonyong Park, Hanjoong Jo2 and Etty N. Benveniste3

Departments of Cell Biology [C. C., X. X., J-W. O., S. J. L., E. N. B.], Pathology [H. P., H. J.], and Surgery [G. Y. G.], University of Alabama at Birmingham, Birmingham, Alabama 35294


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fas transduces not only apoptotic signals through various pathways but also angiogenic and proinflammatory responses in vivo. Human glioma cells express Fas although sensitivity to Fas-mediated cell death is variable, suggesting that Fas may have functions other than apoptosis in these cells. In this study, we addressed alternative functions of Fas expressed on human gliomas by Fas ligation in three human glioma cell lines, CRT-MG, U373-MG, and U87-MG, and the in vivo expression of Fas and chemokines in human glioblastoma multiforme (GBM). Herein, we demonstrate that: (a) stimulation with agonistic anti-Fas monoclonal antibody CH-11 and human recombinant soluble Fas ligand induces expression of the CC chemokine MCP-1 and the CXC chemokine interleukin-8 by human glioma cell lines at the mRNA and protein levels in a dose- and time-dependent manner; (b) selective pharmacological inhibitors of MEK1 (U0126 and PD98059) and p38 mitogen-activated protein kinase (MAPK) (SB202190) suppress Fas-mediated chemokine expression in a dose-dependent manner; (c) Fas ligation on human glioma cells leads to activation of both extracellular signal-regulated kinases ERK1/ERK2 and p38 MAPK; and (d) GBM samples express higher levels of Fas compared with normal control brain, which correlates with increased interleukin 8 expression. These findings indicate that Fas ligation on human glioma cells leads to the selective induction of chemokine expression, which involves the ERK1/ERK2 and p38 MAPK signaling pathways. Therefore, the Fas-Fas ligand system in human brain tumors may be involved not only in apoptotic processes but also in the provocation of angiogenic and proinflammatory responses.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fas/APO-1 (CD95), a member of the tumor necrosis factor/nerve growth factor receptor family, signals apoptotic cell death in susceptible target cells (reviewed in Ref. 1 ). Signals through Fas molecules are mediated by the intracytoplasmic death domain, which associates with the Fas-associated death domain through homeotypic interactions. The Fas-associated death domain can then recruit procaspase-8 to form the death-inducing signaling complex, leading to activation of the downstream caspase cascade and subsequent cell death (2) . The Fas-FasL4 system is one of the effector mechanisms of cytotoxic T cells and natural killer cells and plays an important role in the maintenance of immune-privilege in specialized organs (reviewed in Refs. 3 and 4 ). In the CNS, neurons and some glial cells express FasL and Fas on their cell surfaces and are involved in maintaining the immune-privileged status of the CNS, as well as induction of cell death in pathological conditions such as multiple sclerosis and experimental allergic encephalomyelitis, the animal model for multiple sclerosis (5, 6, 7, 8, 9, 10, 11, 12, 13, 14) .

Recently, angiogenic and proliferative roles for the Fas-FasL system have been suggested (15, 16, 17, 18) , and Fas oligomerization can also induce proinflammatory responses such as the secretion of cytokines and chemokines (IL-1, IL-6, IL-8, and MIP-1{alpha}; Refs. 12 , 19 , and 20 ). In the pancreas of transgenic animals, FasL provokes a granulocytic infiltrate rather than acting as an immunosuppressant (17) . Local stimulation of Fas in vivo by using s.c. implants containing an agonistic anti-Fas mAb induced rapid neovascularization and infiltration of inflammatory cells within the implant (15) .

Tumor cells acquire various defense mechanisms during tumorigenesis to evade the host immune system. As one of the best known mechanisms, tumor cells express functional FasL on their surface, enabling them to induce apoptosis of activated Fas-positive T cells that have infiltrated around the tumor (reviewed in Ref. 4 ). Human gliomas frequently coexpress Fas and FasL (21, 22, 23, 24) . Astroglioma cells express functional FasL that can deliver death signals to Fas-positive infiltrating leukocytes, as well as to the astroglioma cells themselves via Fas expression (22) . However, sensitivity to Fas-mediated cell death in human astroglioma cells is variable (21 , 24 , 25) . Recently, Fas ligation in these cells was shown to result in cell cycle progression via ERK activation (18) , suggesting a role for Fas-FasL in glioma growth regulation. In this study, we examined potential alternative functions of Fas on gliomas that are resistant to Fas-mediated cell death, the involvement of ERK and p38 MAPK pathways in Fas-induced signaling cascades, and the in vivo expression of Fas and IL-8 in human GBM specimens.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Culture Conditions.
CRT-MG and U373-MG human glioma cell lines were maintained in RPMI 1640 with 10 mM HEPES (pH 7.2) and MEM with 1 mM Earle’s BSS medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FBS, as described previously (26) . U87-MG cells were grown in a 50:50 mixture of DMEM and Ham’s F-12 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented as described above.

Patients and Tissue Samples.
Eleven patients with GBM (mean age ± SD, 53.3 ± 11.2 years) and five with temporal lobe epilepsy (mean age ± SD, 33.0 ± 23.2 years) for normal brain cortex were studied. Tumors were graded according to the 1993 WHO classification. It should be noted that all control samples were from temporal lobe neocortex, whereas it is not certain from which normal cells the tumors originated. Tumor biopsy and temporal neocortical specimens were immediately frozen in liquid nitrogen after surgical removal and stored at -80°C. Soluble extracts of the tissues were prepared by crushing in liquid nitrogen and dounce homogenizing with 1 mM phenylmethylsulfonyl fluoride. The supernatants were collected after centrifugation at 4°C for 10 min in a microcentrifuge and stored at -80°C until use.

Reagents.
Mouse monoclonal antihuman Fas IgM antibody CH-11 was obtained from Upstate Biotechnology (Lake Placid, NY). Human recombinant sFasL and cross-linking antibody were purchased from Alexis Biochemicals (San Diego, CA). Mouse monoclonal IgM {kappa} antibody was purchased from Sigma Chemical Co. (St. Louis, MO). Mouse antihuman Fas mAb (IgG1 isotype) conjugated with PE was purchased from PharMingen (San Diego, CA), and mouse IgG1 conjugated to PE was purchased from Southern Biotechnology Associates (Birmingham, AL). The MAPK inhibitors SB202190, PD98059, and U0126 were obtained from Calbiochem (La Jolla, CA), as were the control compounds SB202474 and U0124.

Total RNA Isolation and RPA.
Cells were washed with ice-cold PBS, and then RNA was extracted using a method based on guanidinium isothiocyanate phenol extraction, followed by ethanol precipitation as described previously (26) . A linearized human chemokine multiprobe set (hCK-5; PharMingen) was in vitro transcribed with T7 RNA polymerase, resulting in antisense RNA probes. RPA was carried out as described previously (26) . Four to ten µg of total RNA were hybridized with hCK-5 riboprobes. Values for each chemokine mRNA were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels for each experimental condition.

Flow Cytometric Analysis.
Glioma cell lines (2 x 105 cells/well) were plated in six-well (35-mm2) plates (Costar, Cambridge, MA) and grown to 90% confluency. For analysis of Fas protein expression, U373-MG, CRT-MG, and U87-MG cells were trypsinized, suspended in PBS containing 5% FBS and 0.02% azide, stained with PE-conjugated antihuman Fas antibody (1:2000), washed twice, fixed in 1% paraformaldehyde, and then analyzed on the FACStar (Becton Dickinson, Mountain View, CA). Negative controls were incubated with an isotype-matched (IgG1) control mAb conjugated to PE. Ten thousand cells were analyzed for each sample.

Detection of Apoptosis.
Cell death was determined by staining with Annexin V (PharMingen), a Mr 35,800 protein that has a strong affinity for phosphatidylserine. After treatment with CH-11 or sFasL, cells were washed twice with PBS, trypsinized, suspended in 200 µl of binding buffer, and stained with 0.5 ng of Annexin V-FITC and 2.5 ng PI. Ten thousand cells were analyzed on the FACStar within 30 min after staining. Fractions of cell stained with PI were cells with disintegrated cell membranes, revealing necrosis or late apoptosis (in the case of costaining with Annexin V). Therefore, cell death, including apoptosis and necrosis, was defined as cell fractions stained with Annexin V and/or PI.

Plasmids and Transient Transfection.
The human IL-8 promoter construct used for transient transfection was as described previously (27) , and the pCMV-ß-galactosidase construct was purchased from Clontech (Palo Alto, CA). Transient transfection of the CRT-MG human glioma cell line was performed by electroporation using a Bio-Rad gene pulser as described previously (28) . After 36 h of recovery, cells were incubated in the presence of CH-11 antibody for an additional 24 h. Cells were then harvested, and luciferase and ß-galactosidase activities were measured as described previously (28) . The luciferase activity of each sample was normalized to ß-galactosidase activity to calculate relative luciferase activity.

ELISA.
CRT-MG cells were incubated in the absence or presence of MAPK inhibitors for 1 h, followed by treatment with CH-11 antibody for 24 h in serum-free medium. Concentrations of MCP-1 and IL-8 in the supernatants were assayed using a dual-antibody, solid-phase ELISA for either MCP-1 or IL-8 (Biosource International, Camarillo, CA). The extracts from biopsy specimens were also assayed using ELISA for Fas (PharMingen) as well as IL-8. Measurement of IL-8 was done in duplicate. The lower limits of detection of the ELISAs are 0.1 pg/ml for IL-8 and 0.05 unit/ml for Fas, respectively. The values of IL-8 and Fas protein are normalized to the total protein values of each sample.

Immunoblotting and in Vitro Kinase Assay.
Glioma cells were incubated with CH-11 antibody (500 ng/ml) for various time periods and then harvested. Cell lysates were prepared as described previously (29) , and 100 µg of protein were electrophoresed in 10% SDS gels. Proteins were then transferred to nitrocellulose and probed with rabbit polyclonal antibodies against ERK1/ERK2 (New England Biolabs, Beverly, MA). To analyze the phosphorylated forms of ERK1/ERK2, rabbit polyclonal antibodies specific to phospho-ERK1/ERK2 (Thr-202/Tyr-204) were used (New England Biolabs).

Soluble lysates (100–200 µg) from cultured glioma cells and extracts (100 µg) from biopsy specimens were used to phosphorylate MBP (Sigma) and c-Jun (Calbiochem) as described previously (29) . Lysates were incubated with 1 µg of anti-ERK2, anti-JNK, or anti-p38 MAPK antibody (Santa Cruz) for 1 h at 4°C, followed by an additional 1-h incubation with Protein A/G gel beads (Pierce Corp., Rockford, IL). The immunocomplexes were washed four times in extraction buffer and twice in kinase reaction buffer as described previously (29) . The washed immunocomplexes were incubated in 20 µl of kinase reaction buffer containing 10 µg of MBP or 1 µg of c-Jun and 5.0 µCi of [{gamma}-32P]ATP for 20 min at 30°C. Phosphorylation of MBP or c-Jun was stopped by boiling in Laemmli sample buffer, followed by 10% SDS-PAGE and autoradiography.

Statistical Analysis.
Levels of significance for comparisons between samples were determined using Student’s t test distribution. The Spearman method was used to analyze the correlation between IL-8 and Fas values in the GBM samples.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of Fas and Sensitivity to Fas-mediated Cell Death in Glioma Cells.
We screened Fas expression and sensitivity to Fas-mediated cell death in three human glioma cell lines, CRT-MG, U373-MG, and U87-MG. All glioma cells constitutively express Fas, with varying degrees of expression (Fig. 1A)Citation . Next, the sensitivity of the cells to Fas-mediated apoptosis, induced by the anti-Fas IgM monoclonal antibody CH-11, was assessed. Only U87-MG cells underwent apoptosis (~30%) upon incubation with CH-11 antibody for 24 h in the presence of 10% serum (data not shown). As shown in Fig. 1BCitation , serum starvation augmented Fas-mediated cell death of U87-MG cells (~75%), whereas U373-MG and CRT-MG cells were still resistant to Fas-mediated apoptosis. Because different biological effects have been reported for agonistic anti-Fas antibodies and FasL (24 , 30) , human recombinant sFasL was used to induce Fas-mediated apoptosis. Although the apoptosis-inducing capacity of naturally processed sFasL is reduced compared with membrane-bound FasL, sFasL retains the capacity to interact with Fas, and restoration of its cytotoxic activity is achieved both in vitro and in vivo upon the addition of cross-linking antibodies (30) . Incubation with sFasL (10 ng/ml) in the presence of cross-linking antibody (1 µg/ml) also induced apoptosis in U87-MG cells (~80%) but not in U373-MG and CRT-MG cells (data not shown). IFN-{gamma} treatment has been shown to render cells susceptible to Fas-mediated apoptosis by up-regulation of Fas expression (11 , 20) . Treatment of U373-MG and CRT-MG cells with IFN-{gamma} up-regulated Fas protein expression by ~1.2- and ~1.8-fold, respectively; however, the cells remained nonsusceptible to Fas-mediated apoptosis (data not shown).



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  		Fig. 1. Expression of Fas and susceptibility to Fas-mediated apoptosis in human glioma cells. A, FACS analysis of Fas expression on U373-MG, CRT-MG, and U87-MG cells. ····, negative controls stained with isotype antibody conjugated with PE. B, cells were incubated with various concentrations of CH-11 antibody (0, 20, 100, 200, 500, and 1000 ng/ml) for 24 h in serum-free conditions, and then cell death was measured by staining with Annexin V-FITC and PI. Results are representative of two independent experiments.

 
Fas Ligation of Human Glioma Cells Induces Chemokine Expression.
We wanted to determine whether Fas ligation on the glioma cells resistant to Fas-mediated cell death may have other functions, such as the induction of inflammatory signals. CRT-MG and U373-MG cells were incubated with CH-11 antibody (250 ng/ml) for various time periods (0–10 h), and then RNA was extracted and analyzed by RPA for chemokine mRNA expression. CH-11 antibody treatment lead to the selective induction of IL-8 and MCP-1 mRNA expression in CRT-MG (Fig. 2A)Citation and U373-MG (data not shown) in a time-dependent manner. Optimal IL-8 mRNA expression was observed at 2–4 h after Fas ligation. CH-11-induced MCP-1 mRNA was modest compared with IL-8 mRNA expression; however, MCP-1 mRNA levels peaked at 3 h (Fig. 2A)Citation . Expression of RANTES, MIP-1ß, MIP-1{alpha}, and I-309 was not observed (Fig. 3)Citation . Incubation with 10 ng/ml of sFasL in the presence of 1 µg/ml of cross-linking antibody induced comparable levels of IL-8 and MCP-1 mRNA expression in CRT-MG cells (data not shown). These results collectively indicate that ligation of Fas on human glioma cells resistant to Fas-mediated cell death results in the induction of the chemokines IL-8 and MCP-1, with IL-8 expression being predominant.



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  		Fig. 2. Chemokine induction upon Fas stimulation. A, CRT-MG cells were incubated with CH-11 antibody (250 ng/ml) for various time periods (0–10 h), and RNA was extracted and analyzed by RPA for chemokine mRNA expression. B, U87-MG cells were incubated with CH-11 antibody (250 ng/ml) for up to 48 h, and RNA was prepared and analyzed by RPA. Fold induction of IL-8 and MCP-1 mRNA expression compared with basal levels is indicated. Results are representative of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 


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  		Fig. 3. Dose responsiveness of Fas stimulation on chemokine expression. A, CRT-MG cells were treated with increasing concentrations of isotype antibody or CH-11 antibody for 3 h. RNA was extracted and analyzed by RPA for chemokine mRNA expression. Results are representative of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, quantification of MCP-1 and IL-8 mRNA expression is shown. Values for MCP-1 and IL-8 mRNA from CH-11 antibody treated samples were normalized to the value for control samples, and the fold induction was calculated. C, 20,000,000 CRT-MG cells were transfected with 20 µg of an IL-8 promoter luciferase construct and 20 µg of the pCMV-ß-galactosidase construct as indicated in "Materials and Methods." The cells were incubated with increasing concentrations of CH-11 antibody (0–1000 ng/ml) or 1000 ng/ml of IgM antibody for 24 h, harvested, and then examined for luciferase and ß-galactosidase activities. The luciferase activity of each sample was normalized to the ß-galactosidase activity to calculate relative luciferase activity. Results are representative of two experiments. Each experiment was performed in triplicate, and each sample was analyzed in duplicate. Significantly different from control values: *, P < 0.01 (n = 6). Bars, SE. D, CRT-MG cells were treated with various concentrations of CH-11 antibody ({blacksquare}) and isotype IgM antibody ({square}) for 24 h in serum-free conditions, and the protein expression level of MCP-1 and IL-8 was determined by ELISA. MCP-1 and IL-8 expression was normalized by the amount of total protein. Results are representative of three independent experiments; each experiment was performed in triplicate. Significantly different from control values: *, P < 0.05 (n = 3). Bars, SE.

 
We also examined the potential of U87-MG cells, which are sensitive to Fas-mediated apoptosis, to express chemokine mRNA upon incubation with CH-11 antibody. Such treatment lead to a delayed induction of IL-8 mRNA at 12–18 h, which continued to increase up to 48 h (Fig. 2B)Citation . MCP-1 mRNA expression was not significantly affected. Although the incubation with CH-11 antibody was done in the presence of 10% FBS, a considerable number of U87-MG cells started to undergo apoptosis at ~36 h (40% cell death); thus, stimulation with CH-11 was not extended past 48 h.

For a more extensive analysis of chemokine expression upon Fas ligation, we used the CRT-MG cells because they expressed the highest levels of chemokine mRNA. CRT-MG cells were incubated with increasing concentrations of CH-11 antibody or an isotype-matched control antibody (0–1000 ng/ml) for 3 h, and then RNA was analyzed by RPA. Incubation with an IgM control antibody had no effect on chemokine mRNA expression (Fig. 3ACitation , Lanes 2–6). CH-11 antibody treatment induced both MCP-1 and IL-8 mRNA expression in a dose-dependent manner, with optimal induction observed using 500-1000 ng/ml of antibody (Fig. 3ACitation , Lanes 7–11). An ~7.5-fold enhancement of MCP-1 mRNA levels was detected upon stimulation with 500 ng/ml of CH-11 antibody (Fig. 3B)Citation , whereas IL-8 mRNA levels increased by ~40-fold at the highest concentration of antibody tested (1000 ng/ml; Fig. 3BCitation ). Of interest, IP-10 mRNA was modestly induced upon stimulation of CRT-MG cells with 500-1000 ng/ml of CH-11 antibody (Fig. 3ACitation , Lanes 10 and 11). In U373-MG and U87-MG cells, even high concentrations of antibody (1000 ng/ml) did not induce IP-10 mRNA expression at the 3-h time point (data not shown).

To determine whether Fas ligation affected chemokine expression at the transcriptional level, IL-8 promoter activity was tested in CRT-MG cells. The human IL-8 promoter consists of 546 bp including activator protein-1, nuclear factor-{kappa}B, and nuclear factor-IL-6-responsive elements (27) . CRT-MG cells were transfected with the IL-8 promoter construct and a pCMV-ß-galactosidase construct to monitor transfection efficiency and then incubated with increasing concentrations of CH-11 antibody (0–1000 ng/ml) for 24 h. Fas ligation induced luciferase reporter activity in a dose-dependent manner (~5-fold induction at 1000 ng/ml), whereas incubation with an isotype IgM antibody (1000 ng/ml) had no effect (Fig. 3C)Citation .

We next examined parameters of chemokine protein expression upon Fas ligation. CRT-MG cells were incubated with increasing concentrations of the CH-11 antibody or IgM isotype control (0–1000 ng/ml) for 24 h, and then supernatants were harvested and analyzed by ELISA for MCP-1 and IL-8 production. As shown in Fig. 3DCitation , both MCP-1 and IL-8 protein production occurred in a dose-dependent manner upon incubation with CH-11 antibody, whereas the isotype control antibody was without effect. Optimal induction of both chemokines was observed using the CH-11 antibody at a concentration of 1000 ng/ml. Similar findings were observed in U373-MG cells (data not shown).

Signal Transduction Pathways Mediating Fas Induction of Chemokine Expression.
To begin to elucidate the signal transduction cascades initiated upon Fas ligation that are responsible for chemokine induction, we used a variety of pharmacological inhibitors of the MAPK signaling cascade. We tested specific inhibitors of MAPK: SB202190, a selective p38 MAPK inhibitor (31) ; and U0126 and PD98059, selective inhibitors of MEK1 and the downstream MAPKs, ERK1 and ERK2 (32 , 33) . Pretreatment of cells with SB202190 caused a dose-dependent inhibition of CH-11 antibody-induced MCP-1 and IL-8 mRNA expression (Fig. 4ACitation , Lanes 6–10). As well, incubation with the MEK1 inhibitor U0126 resulted in a strong suppressive effect on CH-11 antibody-induced MCP-1 and IL-8 mRNA expression (Fig. 4BCitation , Lanes 6–10), whereas U0124, a negative control for U0126, had no effect on Fas-mediated chemokine mRNA expression (Fig. 4BCitation , Lanes 11 and 12). The MEK1 inhibitor PD98059 showed a similar pattern of inhibition of Fas-mediated chemokine mRNA expression, as did the U0126 MEK1 inhibitor (data not shown). Preincubation of cells with both SB202190 and U0126 resulted in an additive inhibitory effect on IL-8 and MCP-1 mRNA expression (Fig. 4CCitation , Lanes 5–8). These results indicate that Fas-mediated stimulation of MCP-1 and IL-8 mRNA expression involves activation of ERK1/ERK2, as well as p38 MAPK. Comparable results were obtained when examining the influence of SB202190 and U0126 on Fas-induced IL-8 protein production (Fig. 4D)Citation .



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  		Fig. 4. MEK1 and p38 MAPK inhibitors suppress Fas-mediated chemokine expression. CRT-MG cells were treated with various MAPK inhibitors for 1 h prior to a 3-h treatment with CH-11 antibody (500 ng/ml), and chemokine mRNA expression was examined by RPA (A–C). Fold induction of IL-8 and MCP-1 mRNA expression is indicated. SB202190 was used at concentrations of 0.01–10 µM in the absence or presence of CH-11 antibody (A). U0126 was tested at concentrations of 0.01–10 µM in the absence or presence of CH-11, and the negative control for U0126, U0124, was used at a concentration of 10 µM (B). CRT-MG cells were pretreated with SB202190 (10 µM) and U0126 (10 µM) alone or in combination and then were incubated in the absence or presence of 500 ng/ml of CH-11 antibody for 3 h. Results are representative of three independent experiments (C). CRT-MG cells were incubated in the absence or presence of SB202190 (10 µM) and/or U0126 (10 µM) for 1 h and treated with 500 ng/ml of CH-11 antibody for 24 h in serum-free conditions; then supernatants were harvested and analyzed for IL-8 protein by ELISA (D). Results are representative of two independent experiments. Significantly different from control values: *, P <= 0.002. IL-8 protein values from samples pretreated with various MAPK inhibitors were significantly different from the values from the CH-11-treated sample without inhibitors: **, P <= 0.001. Bars, SE. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
Our results demonstrated an involvement of ERK1/ERK2 and p38 MAPK in mediating Fas-induced signals leading to chemokine expression. Thus, we examined the status of MAPK activation upon Fas ligation. As shown in Fig. 5ACitation , treatment of cells with the CH-11 antibody led to a time-dependent increase in ERK1/ERK2 phosphorylation, with optimal phosphorylation observed between 60 and 90 min (Lanes 5 and 6). IL-1ß (5 ng/ml) treatment for 60 min was included as a positive control (Fig. 5ACitation , Lane 8). Total ERK1/ERK2 protein levels are shown in the lower panel. Similarly, the same extracts were examined for JNK/SAPK phosphorylation, and no effect was noted over the 2-h time course (data not shown). An in vitro kinase assay for JNK/SAPK also revealed no kinase activity upon treatment with CH-11 antibody (data not shown). Treatment with the CH-11 antibody induced a time-dependent increase of p38 MAPK activity, with optimal in vitro kinase activity observed at 30 min (Fig. 5B)Citation . Pretreatment of cells with the MAPK inhibitors U0126 and SB202190 abrogated CH-11 antibody induced ERK2 and p38 MAPK in vitro activities, respectively (Fig. 5C)Citation . Interestingly, the MEK inhibitor U0126 also reduced Fas-induced p38 MAPK activity (Fig. 5CCitation , Lane 3). These results demonstrate that Fas ligation on glioma cells leads to activation of two MAPKs, ERK1/ERK2 and p38 MAPK.



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  		Fig. 5. Fas ligation activates ERK1/ERK2 and p38 MAPK. A, cells were incubated with CH-11 (250 ng/ml) for various time periods (0–2 h), cell lysates were prepared, and then 100 µg of protein were electrophoresed in 10% SDS gels. Proteins were transferred to nitrocellulose and probed with antibody specific to phosphorylated ERK1/ERK2 (1:1000; top). IL-1ß (4 ng/ml) treatment for 60 min was included as a positive control (Lane 8). Total ERK1/ERK2 protein levels are shown (bottom). B, in vitro kinase activity of p38 MAPK was measured after immunoprecipitation with anti-p38 MAPK antibody (top). Bottom, total p38 MAPK protein levels by Western blotting with anti-p38 MAPK antibody. Results are representative of four independent assays. C, cells were incubated in the absence or presence of U0126 (10 µM) or SB202190 (10 µM) for 1 h and treated with 500 ng/ml of CH-11 antibody for 45 min, and the in vitro kinase activities of ERK2 and p38 MAPK were measured. Results are representative of two independent assays.

 
In Vivo Expression of IL-8 and Fas in Human GBMs.
To determine in vivo levels of IL-8 and Fas expression, extracts from tumor biopsy and normal brain cortex from epilepsy surgery specimens were analyzed by ELISA for IL-8 and Fas. As shown in Table 1Citation , both Fas and IL-8 protein levels were increased in GBM samples compared with control samples. Fas protein was constitutively expressed in normal brain (mean ± SE, 22.3 ± 3.3 units/mg protein) and significantly increased in GBM (51.7 ± 7.2 units/mg protein; P <= 0.05). IL-8 protein was not detectable in the five normal brain samples, whereas IL-8 was detected in 7 of 11 GBM samples (139.8 ± 97.6 pg/mg protein). The levels of IL-8 correlated significantly with the levels of Fas (Spearman’s correlation, R = 0.67, P <= 0.01). These results collectively indicate that GBM samples express significantly higher levels of Fas protein compared with normal brain, which correlates with increased IL-8 expression.


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  		Table 1 In vivo expression of Fas and IL-8 by human GBM

 
Because we have shown that the activation of MAPK is responsible for Fas-mediated chemokine expression in established human glioma cell lines, we next examined the status of ERK and p38 MAPK activation in human brain specimens. We performed the in vitro kinase assay for ERK2 and p38 MAPK using extracts (100 µg of total protein) from the 11 GBM samples and 5 normal controls. The GBM samples had highly variable levels of both p38 and ERK MAPK activities; thus, MAPK activity was not significantly increased in GBM samples compared with normal controls (data not shown).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we observed that some human glioma cells, despite abundant Fas expression, are resistant to Fas-mediated apoptosis, raising the question as to whether the Fas molecule has functions other than apoptosis. The results presented herein demonstrate that Fas ligation results in chemokine (IL-8 and MCP-1) expression in human glioma cells through MAPK signaling cascades. Fas ligation was achieved in two ways: one by the use of the agonistic anti-Fas antibody CH-11; and the other using the soluble form of the natural ligand FasL (sFasL) and a cross-linking antibody. Comparable results were achieved with both reagents. FasL expression has been shown to be common in human astrocytic brain tumors, and expression is localized to the plasma membrane of glioma cells (22 , 23 , 34 , 35) . Furthermore, FasL is functional in that FasL-expressing astroglioma cells (both established cell lines and tumor cells directly obtained after enzymatic dissociation of tumor biopsy), efficiently and specifically killed Fas-positive target cells (22) . Infiltrating activated T cells also serve as an in vivo source of FasL (36) . Thus, within tumor samples, functional FasL is present and potentially capable of mediating chemokine expression in vivo.

IL-8, a member of the CXC family of chemokines, is a potent chemoattractant and activator of neutrophils and has angiogenic properties (37 , 38) . IL-8 is produced by most astroglioma and glioblastoma cell lines upon incubation with various stimuli such as lipopolysaccharide, IL-1ß, and tumor necrosis factor-{alpha} (26) . Furthermore, IL-8 is up-regulated in glioma cell lines in response to ischemia/hypoxic conditions (38 , 39) . Our results confirm previous data showing that IL-8 expression is elevated in GBM (39) , and we now show that this expression correlates with increased levels of Fas. The contribution of IL-8 in CNS tumorigenesis is unclear at this time. Although IL-8 is a chemoattractant for neutrophils, these cells are rarely seen in glial tumors (39) . Rather, IL-8 may influence angiogenesis by affecting microvascular endothelial cell morphogenesis (38) ; as such, it is likely that IL-8 may promote tumor cell growth (40) . A recently proposed function for IL-8 is as a suppressor of Fas-mediated apoptosis of human fetal astrocytes (12) . In this report, late-passage astrocytes were sensitive to Fas ligation and subsequent apoptosis, whereas early-passage astrocytes were totally resistant to Fas-mediated apoptosis. An increase in IL-8 secretion occurred after Fas ligation on early-passage astrocytes, which correlated with the ability of the astrocytes to resist Fas-mediated apoptosis (12) . However, our preliminary results indicate that MAPK inhibitors, which suppress IL-8 expression, do not potentiate or sensitize human astroglioma cell lines (U373-MG and CRT-MG) to Fas-mediated apoptosis (data not shown). Furthermore, one subclone of CRT-MG cells, which has lost the ability to activate p38 MAPK upon Fas ligation and subsequently cannot induce IL-8 expression, is also resistant to Fas-mediated apoptosis (data not shown). Additionally, we have shown that IL-8 is induced in the apoptosis-sensitive cell line U87-MG upon Fas ligation, albeit with delayed kinetics. Therefore, the antiapoptotic effect of IL-8 may be restricted to fetal astrocytes, perhaps because of different environmental cues (12) .

MCP-1, a member of the CC chemokine family, attracts monocytes, memory T cells, and natural killer cells (reviewed in Ref. 41 ) and is expressed by human astrogliomas in vivo and in vitro (42 , 43) . Infiltrating macrophages are commonly found in human gliomas, and the degree of macrophage infiltration correlates with the level of MCP-1 mRNA and protein expression (43) . Although the function of the infiltrating macrophages has not been determined, MCP-1 has been considered as a potential chemokine to enhance antitumoral effects within the tumor by increasing the number of macrophages within this site. Fas ligation on glioma cells may serve as a mechanism to enhance MCP-1 expression and subsequent macrophage recruitment.

Another member of the CXC chemokine family, IP-10, plays an important role in T-cell recruitment and has angiostatic properties (44 , 45) . Because IP-10 was only induced upon stimulation with high concentrations of CH-11 (>=500 ng/ml) in one human astroglioma cell line, CRT-MG, the physiological relevance of IP-10 in brain tumors is unknown at this time. Interestingly, induction of IP-10 was exclusively inhibited by the p38 MAPK inhibitor (data not shown), suggesting that the signal transduction pathway may be different from those for IL-8 and MCP-1 induction.

MAPKs are serine-threonine protein kinases that are activated by diverse stimuli including cytokines, growth factors, neurotransmitters, hormones, cellular stress, and cell adhesion (reviewed in Ref. 46 ). The basic assembly of the MAPK pathway is a three-component module and includes three kinases that establish a sequential activation pathway consisting of a MAPK kinase kinase, MAPK kinase, and MAPK. More than a dozen mammalian MAPK family members have been discovered, which include the ERK1/ERK2, p38 MAPK, and JNK/SAPK pathways (reviewed in Refs. 46 and 47 ). We have shown that ERK1/ERK2 and p38 MAPK, but not JNK/SAPK, are activated upon Fas ligation in glioma cells, and pharmacological inhibitors of ERK1/ERK2 and p38 MAPK block Fas-mediated induction of chemokine expression. Recently, Shinohara et al., (18) reported that Fas ligation induces ERK activation and subsequently drives cell cycle progression in human glioma cells; however, they could not detect activation of p38 MAPK. We have used a more sensitive in vitro kinase assay method to detect p38 MAPK activity; with conventional immunoblot methodology, we also could not detect p38 MAPK activation upon Fas ligation in human gliomas. However, we did not detect an increase in in vivo p38 or ERK MAPK activity in the GBM samples compared with normal brain specimens. Activated MAPK are rapidly dephosphorylated and subsequently inactivated by phosphatases, and this rapid process enables the temporal activation of MAPK signal transduction pathways (reviewed in Ref. 46 ). Therefore, although MAPK activation is important for IL-8 and MCP-1 expression in vitro, the activity of MAPKs may not be sustained in vivo. However, it is still possible that tumor cells may use different signal transduction pathways other than MAPK pathways in vivo. It will be important to determine the in vivo roles of MAPKs in chemokine induction and subsequent biological responses in brain tumor models.

It is not yet known which components of the Fas downstream signaling cascades leads to activation of ERK1/ERK2 and p38 MAPKs. The intracytoplasmic death domain of Fas recruits several adaptor molecules to activate downstream transducers. One of these, daxx, activates apoptosis signaling kinase 1 and subsequently JNK kinase and JNK (48) . Although human glioma cells express daxx mRNA (data not shown), JNK activation is not induced upon Fas ligation in CRT-MG cells. However, we cannot exclude the possibility that daxx may transduce MAPK signaling via an alternative pathway in glioma cells. The more proximal domain to the death domain of Fas also transduces signals through the activation of acidic and neutral sphingomyelinases (49 , 50) . The small G proteins rac-1 and ras have also been reported to be involved in Fas-mediated apoptosis (51) , although their roles in Fas-mediated signals other than apoptosis have not been determined. Therefore, the poorly defined proximal portion of the Fas molecule may activate membrane-bound small G proteins and the ceramide pathway to ultimately activate MAPK pathways, resulting in chemokine expression upon Fas ligation. It has been shown recently that blockage of caspase activation abrogated Fas-mediated MAPK activation in human glioma cells, suggesting that caspases may be upstream of the MAPK pathway in human gliomas (18) . These possibilities are under investigation in our laboratory.

The biological functions of the Fas-FasL system in brain tumors are complex (Fig. 6)Citation : (a) the expression of FasL by malignant astrocytomas, the most common and malignant primary brain tumor, is a mechanism by which these cells evade the host immune system by inducing apoptosis of Fas-positive T cells that infiltrate into the tumor tissue (Fig. 6ACitation ; Refs. 22 and 52 ); and (b) Fas ligation on astroglioma cells can provoke apoptotic (21 , 24) and/or proliferative (18) responses (Fig. 6B)Citation . We have shown in this report that Fas ligation on these cells also induces inflammatory mediators such as chemokines (Fig. 6B)Citation . In vivo CNS sources of FasL include infiltrating T cells and astroglioma cells themselves. Under these circumstances, Fas-mediated signals may be regarded as targets for suppression of proliferation and/or inflammatory responses in CNS tumors. Elucidation of the molecular mechanisms responsible for the inflammatory functions of the Fas-FasL system in brain tumors will be important for a better understanding of glioma biology.



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  		Fig. 6. Roles of the Fas-FasL system in the brain tumors. A, FasL expressed in the CNS is involved in maintenance of immune privilege or evasion from the host immune system by tumor cells. B, activated T cells and astroglioma cells expressing FasL can induce apoptotic cell death of susceptible astroglioma cells through Fas-FasL interactions as well as proliferative and proinflammatory responses.

 


   ACKNOWLEDGMENTS
 
We thank Dr. Naofumi Mukaida (Kanazawa University, Kanazawa, Japan) for the human IL-8 promoter construct. We acknowledge the support of the University of Alabama at Birmingham Flow Cytometry Core Facility (Grant AM20614).


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grants MH55795, NS36765, and NS29719 (to E. N. B.). Back

2 Present address: Georgia Tech/Emory Biomedical Engineering, Emory University, Atlanta, GA 30322. Back

3 To whom requests for reprints should be addressed, at Department of Cell Biology, MCLM 395, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, AL 35294-0005. Phone: (205) 934-7667; Fax: (205) 975-6748; E-mail: tika{at}uab.edu Back

4 The abbreviations used are: FasL, Fas ligand; sFasL, soluble FasL; CNS, central nervous system; IL, interleukin; MIP, macrophage inflammatory protein; mAb, monoclonal antibody; PE, phycoerythrin; RPA, RNase protection assay; PI, propidium iodide; CMV, cytomegalovirus; ERK, extracellular signal-regulated kinase; MBP, myelin basic protein; GBM, glioblastoma multiforme; JNK/SAPK, c-Jun NH2-terminal kinase/stress-activated protein kinase; MAPK, mitogen-activated protein kinase. Back

Received 9/ 7/00. Accepted 1/26/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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