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7,8-Dihydroneopterin induces apoptosis of Jurkat T-lymphocytes via a Bcl-2-sensitive pathway
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European Journal of Cell Biology 81, 197 ± 202 (2002, April) ¥ ¹ Urban & Fischer Verlag ¥ Jena http://www.urbanfischer.de/journals/ejcb
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7,8-Dihydroneopterin induces apoptosis of Jurkat T-lymphocytes via a Bcl-2-sensitive pathway Christiane Enzingera, Barbara Wirleitnera, Christina Lutzb, G¸nther Bˆckc, Bettina Tomasellia, Gottfried Baierb, Dietmar Fuchsa, Gabriele Baier-Bitterlich1)a a
b c
Institute for Medical Chemistry and Biochemistry and Ludwig Boltzmann Institute for AIDS Research, Innsbruck/Austria Institute for Medical Biology and Human Genetics, Innsbruck/Austria Institute for Experimental Pathology, Innsbruck/Austria
Received August 20, 2001 Received in revised version December 19, 2001 Accepted December 20, 2001
Apoptosis ± pteridines ± oxidative stress ± CrmA ± Bcl-2 ± Jurkat T-lymphocytes ± 7,8-dihydroneopterin Activated cell-mediated immunity is known to be accompanied by elevated concentrations of 7,8-dihydroneopterin which in high concentrations was found to interfere with the oxidantantioxidant balance. In this study we investigated whether 7,8dihydroneopterin mediates apoptosis of Jurkat T-lymphocytes via a CrmA- or Bcl-2-sensitive pathway. Transient transfection assays with CrmA and Bcl-2 expression constructs showed that apoptosis was not affected by CrmAwhereas it was significantly decreased upon cotransfection with Bcl-2 constructs. Results suggest that 7,8-dihydroneopterin-induced apoptosis of Tlymphocytes is mediated by a Bcl-2-sensitive pathway.
Introduction Neopterin and 7,8-dihydroneopterin are synthesized in human macrophages upon induction with IFN-g (Huber et al., 1984; Bitterlich et al., 1988; Fuchs et al., 1988; Werner et al., 1989). In vivo, the production of neopterin closely correlates with IFN-g concentrations and the activation of cell-mediated immunity, e.g. in human immunodeficiency virus (HIV) infection, neopterin levels increase in parallel with progression of the disease (Fuchs et al., 1988, 1989). In vitro, the excretion of neopterin was found to correlate well with hydrogen peroxide levels (Nathan, 1986), and may therefore serve as a marker for oxidative stress in disease (Fuchs et al., 1997; Murr et al., 1999). By measurement of hydrogen peroxide-mediated luminol chemiluminescence, which allows indirect quantification of 1)
Professor Dr. Gabriele Baier-Bitterlich, Institute for Medical Chemistry and Biochemistry, Fritz Pregl Str. 3, A-6020 Innsbruck/ Austria, e-mail: [email protected], Fax: 43 512 507 2865.
reactive oxygen intermediates, a bimodal effect of 7,8-dihydroneopterin on hydrogen peroxide-mediated chemiluminescence was found. Lower concentrations scavenged, but at high doses (5 mM) 7,8-dihydroneopterin enhanced chemiluminescence (Weiss et al., 1993, Murr et al., 1994, 1996; BaierBitterlich et al., 1995; Reibnegger et al., 1995). This latter observation concedes with data in the literature (Blair and Pearson, 1973) that reduced pteridines react with molecular oxygen under formation of free radicals. At low concentrations it seems that 7,8-dihydroneopterin simply reacts as a chemical reductant destroying reactive oxygen intermediates. In further investigations, electron spin resonance measurements revealed that 7,8-dihydroneopterin in combination with the spin trap DMPO induces the production of DMPO-OH spin adducts, suggesting that 7,8-dihydroneopterin may give rise to the formation of OH radicals via generation of superoxide anion (Oettl et al., 1999; Wirleitner et al., 2001). In later experiments 7,8-dihydroneopterin was shown to function as an electron source needed to reduce dioxygen which led to the hypothesis that human monocytes/macrophages may strongly enhance their aggressive potential via the radical- generating capability of 7,8-dihydroneopterin in the presence of dioxygen (Reibnegger et al., 2001). Interestingly, neopterin and 7,8-dihydroneopterin were indeed found to interfere with redox-sensitive intracellular signaling pathways, and results implied a potential role of neopterin-derivatives in oxidative stress-mediated apoptosis (Fuchs et al., 1994a; Baier-Bitterlich et al., 1995, 1996a, b; Schobersberger et al., 1996; Wirleitner et al., 1998; Spˆttl et al., 2000). Upon incubation with high doses of 7,8dihydroneopterin, Jurkat T-lymphocytes revealed increased apoptosis by measurement of the intranuclear contents of fluorescing DNA by propidium iodide staining and FACS analysis (Baier-Bitterlich et al., 1996c). In order to study the potential modulatory effects of pteridines on mitochondrial function, isolated rat brain mitochondria were exposed to neopterin and 7,8-dihydroneopterin. Whereas exposure of
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mitochondria to either neopterin or 7,8-dihydroneopterin showed no effect on mitochondrial electron transport chain complex I, complex IV and citrate synthase activities, both compounds inhibited complex II/III in a dose-dependent manner (Wede and Fuchs, 1997). The effect of pharmacological inhibitors on 7,8-dihydroneopterin-mediated apoptosis was studied. While apoptosis was not inhibited by a broad range of pharmacological inhibitors such as actinomycin D, cycloheximide, cyclosporin A and various protein kinase inhibitors, inhibitors with antioxidant abilities such as pyrrolidinedithiocarbamate Nacetylcysteine (NAC), superoxid dismutase (SOD) and catalase (CAT) significantly blocked 7,8-dihydroneopterin-mediated apoptosis (Baier-Bitterlich et al., 1996c; Wirleitner et al., 1998). Reactive oxygen intermediates play an important role in cellular metabolic events such as signal transduction and regulation of gene expression (Meyer et al., 1994). As a consequence of oxidative stress a number of early response genes such as c-fos, c-myc and c-jun are transcriptionally activated (Crawford et al., 1988; Datta et al., 1992). Along this line it was observed that incubation of cells with neopterin or 7,8-dihydroneopterin induces c-fos gene expression (‹berall et al., 1994) and leads in conjunction with TNF-a to synergistic activation of NF-kB (Baier-Bitterlich et al., 1997a), which is also a well known oxidative stress responsive transcription factor (Schreck et al., 1991). Apoptosis induced by different stimuli in the same cell may be regulated by different pathways which are often CrmAsensitive or CrmA-resistant. While CrmA-sensitive pathways are frequently Bcl-2/Bcl-XL-resistant, the latter are Bcl-2/BclXL-sensitive (Cohen, 1997). Recently the oncogene Bcl-2 has attracted attention since its control function over apoptosis commitment in disease development and clinical response to therapies has been targeted for pharmacological intervention (for a review see (Voehringer and Meyn, 2000)). In this paper, we intended to investigate whether 7,8-dihydroneopterininduced apoptosis of T lymphocytes is mediated by Bcl-2- or CrmA-sensitive pathways. As a model system Jurkat Tlymphocytes were chosen which were transiently transfected with CrmA and Bcl-2 promoter-reporter gene constructs.
Material and methods Cell culture
CD4 Jurkat Tag cells (a kind gift from Dr. G. Crabtree, Stanford University, CA) which stably express the SV40-derived large T antigen were cultivated in RPMI-1640 medium (Biochrom, Berlin, FRG) complemented with 2 mM glutamine (Serva, FRG), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Biological Industries, Israel) and 10% heat-inactivated fetal calf serum (FCS, Biochrom, Berlin, FRG).
Reagents and plasmids 7,8-Dihydroneopterin was purchased from Schircks Lab. (Jona, Switzerland). The pEF-neo CrmA and Bcl-2 plasmids were a kind gift from Dr. R. Kofler (University of Innsbruck, Austria). The expression plasmid encoding the murine MHC class I protein H2-Kk was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Staurosporine was purchased from Sigma (Vienna, Austria).
Transfection of Jurkat T-cells to study the influence of Bcl-2 and Crm-A Jurkat Tag cells were double-transfected by electroporation with the indicated pEF-neo empty vector control, Bcl-2 or CrmA expression plasmids and the surface marker expression plasmid encoding the murine MHC class I H2-Kk, respectively, as follows. 24 hours after medium change JurkatTag cells were harvested by centrifugation and washed twice in medium without supplements at a concentration of 2 107 cells/400 ml. Aliquots of cell suspensions were transferred to Eppendorf vials and mixed with 20 mg plasmid DNA (neo, Bcl-2 or CrmA). To control for transfection efficiency, 10 mg of the control plasmid H2-Kk were subsequently added. Cells were transferred to the electroporation cuvettes, incubated for 10 min on ice and electroporated in a BTX-Electroporator T820 at 450 kV, 5 pulses, each 99 msec/3.0 kV. After electroporation, cells were incubated for 10 min at room temperature, resuspended in medium with supplements, seeded in culture plates and incubated for 48 hours. During this time cells (1 106) were stimulated with staurosporine (1 mM) or 7,8-dihydroneopterin (5 mM) for 6, 24 or 48 hours.
Detection of apoptotic cells Rates of apoptosis in transfected Jurkat cells were determined by flow cytometric evaluation of annexin-positive cells within the MHC I H2Kk-positive population 48 h after transfection. Cells were harvested and washed once in KR-buffer (Krebs Ringer-buffer; 145 mM NaCl, 5 mM KCl, 3 mM CaCl2, 10 mM glucose, 1.2 mM NaH2PO4, 1 mM MgSO4 and 20 mM Hepes), incubated in 200 ml KR-buffer containing phycoerythrin-labelled anti-mouse H2-Kk monoclonal antibody (1 ml/200 ml KRbuffer; Pharmingen, San Diego, CA, USA) to identify transfected cells and FITC-labelled annexin V (1 ml/200 ml KR-buffer; Alexis Biochemicals) for 30 min on ice. Finally, cells were washed, resuspended in 200 ml annexin binding buffer and analyzed by FACS-Scan. In parallel, cells were stained with Hoechst 33342 (Molecular probes, OR, USA) and analysed on an Olympus BX50 fluorescence microscope to characterize apoptotic nuclei.
Activation of p42/p44 mitogen-acivated protein kinase (MAP kinase) After electroporation cells were incubated for 10 min at room temperature, resuspended in medium with supplements, seeded in culture plates and incubated for 48 hours. During this time cells (1 106) were stimulated with 7,8-dihydroneopterin (5 mM) for 6 and 24 hours. To prepare cell lysates, cells were solubilised in 2 lysis buffer (100 mM Tris-HCl, pH 8.5, 2% NP-40, 10 mM EDTA, 10 mM NaF, 10 mM NaCl, 100 mM Na3VO4, 1 mg/ml aprotinin, 1 mg/ml leupeptin) and incubated for 20 min on ice. Following centrifugation (13 000g, 15 min, 4 8C), supernatants were analysed for protein content (Bradford protein assay, Bio-Rad, Vienna, Austria). 30 mg cell lysate protein were separated on an SDS polyacrylamide gel (12%) and transferred to nitrocellulose membranes (Bio-Rad). Immunoblotting was performed with anti-active MAP kinase antibodies (New England Biolabs, Inc., USA). Phosphorylated MAP kinase was detected with the ECL system (Amersham Pharmacia Biotech, UK). Values are representative of 2 (CrmA) to 3 experiments.
Results and discussion The induction of pteridine-mediated apoptosis of transfected Jurkat Tag cells was first analysed by Hoechst 33342 staining. A significant increase in apoptosis was induced by 7,8-dihydroneopterin (5 mM). Characteristics typical of apoptosis (e.g. chromatin condensation and formation of apoptotic bodies) were observed in transfected cells that were incubated with 7,8dihydroneopterin. Staurosporine was used as a positive control. (Fig. 1A ± D). Data correspond well with earlier findings in various other cell types (Baier-Bitterlich et al., 1995; Schobers-
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Fig. 1. 7,8-Dihydroneopterin-mediated apoptosis of transfected Jurkat T-lymphocytes. Jurkat Tag cells transfected with the control plasmid (PEF-neo) were incubated for 6 ± 24 hours with 7,8-dihydroneopterin (5 mM ) or staurosporine (1 mM) and stained with Hoechst 33342. Cells were analyzed on an Olympus BX50 fluorescence microscope (100 oil
immersion objective) to characterize apoptotic nuclei. (A) untreated control, (B) staurosporine (6 h), (C) 7,8-dihydroneopterin (6 h), (D) 7,8-dihydroneopterin (24 h). Micrographs are representative of 3 experiments.
berger et al., 1996; Spˆttl et al., 2000), e.g. neopterin and 7,8dihydroneopterin in conjunction with TNF-a were shown to intervene with the ability of U937 cells to maintain an appropriate oxidant-antioxidant balance. While concentrations of neopterin up to 1 mM and 7,8-dihydroneopterin up to 300 mM decreased TNF-induced apoptosis, higher doses of 7,8dihydroneopterin (1 mM) did not alter, and at a concentration of 5 mM even superinduced TNF-a-mediated apoptosis. Serum concentrations usually range from 5 nM in healthy individuals to 300 nM neopterin and to 1 mM 7,8-dihydroneopterin in severely ill patients (Fuchs et al., 1994b). Thus, the effective concentrations of 7,8-dihydroneopterin used in our cell culture approach appear to be unphysiological. Yet, in contrast to an only once added dose of stimulants in our experiments, a continuously high level of neopterin and 7,8dihydroneopterin persists in body fluids of patients due to continuous stimulation with IFN-g. Direct cell-to-cell contact may further facilitate the accumulation of even higher concentrations of 7,8-dihydroneopterin in the microenvironment of cells, and its effects may be further augmented in the concert of other inflammatory cytokines which are induced upon activation of cells with IFN-g (Billiau and Dijkmans, 1990) such as interleukin-1 and TNF-a.
Next the effect of CrmA and Bcl-2 on 7,8-dihydroneopterinmediated apoptosis was analysed. In control experiments cells were treated with staurosporine. As expected from recent data in the literature, Bcl-2 significantly decreased staurosporineinduced apoptosis after 24 hours while the inhibition by CrmA was less pronounced (e.g. (Yamashita et al., 2001; Mathiasen et al., 1999; Cohen, 1997)) (Fig. 2A). In contrast 7,8-dihydroneopterin-induced apoptosis was only inhibited in cells which were transfected with the Bcl-2 expression plasmid (Fig. 2B). Data fit well with investigations on the effect of 7,8-dihydroneopterin on MAP kinase (Fig. 3). The impact of 7,8-dihydroneopterin on the activity of the −survival ± associated× MAP kinase, in Jurkat cells transfected with CrmA or Bcl-2 expression plasmids was studied. MAP kinase activity in control cells and in CrmA-transfected cells had ceased after 24 hours but was still significantly elevated in cells expressing Bcl-2. This result again supports the previous observation of the impact of Bcl-2 expression on 7,8-dihydroneopterin-mediated apoptosis. The underlying molecular mechanism, however, remains to be elucidated. CrmA prevents apoptosis in a number of different systems, however its inhibiting capability is dependent on the stimulus to induce it. Apparently there are CrmA-sensitive and -resistant pathways present in the same cell type. CrmA is a
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Fig. 2. Effect of CrmA and Bcl-2 on (A) staurosporine- and (B) 7,8-dihydroneopterin-mediated apoptosis of Jurkat T-lymphocytes. Jurkat Tag cells (2 107 per experiment) were transfected with 20 mg plasmid DNA (pEF-neo, CrmA or Bcl-2) and 10 mg of the −transfection control− plasmid (H2-Kk -MHC I). After electroporation and a resting period cells were seeded in culture dishes and incubated for 48 hours. During this time cells (1 106) were stimulated with (A) staurosporine (1 mM) or (B) 7,8-dihydroneopterin (5 mM) for 6, 24 or 48 hours. Transfected cells were harvested, washed and incubated with phycoerythrin-labelled anti-mouse H2-Kk monoclonal antibody and FITC-labelled annexin V. Cells were analysed on a FACS SCAN. Values were expressed as fold of control (apoptosis (%): control pEF-neo 11.87 1.80; control Bcl-2 13.54 2.16; control CrmA 12.06 1.50) and represent the mean and SEM of 4 experiments. * p < 0.05.
poor inhibitor of CED-3, caspase-2, -3, -7, and -10, but an effective inhibitor of caspase-1, -4, -6 and -8 (see review by Cohen (1997)). Interestingly, many of the apoptotic pathways that are apparently sensitive to inhibition by CrmA are releatively resistant to Bcl-2/Bcl-XL, and vice versa (compare
Fig. 3. Activation of MAP kinase in transfected Jurkat T-cells. p42/44 MAP kinase activation was compared by immunoblotting in 7,8dihydroneopterin-treated Jurkat tag cells transfected with pEF-neo, CrmA or Bcl-2 expression constructs. Cells were pelleted and resuspended in lysis buffer. Cell lysates were cleared by centrifugation and equal amounts of protein were separated by SDS-PAGE. Blots were incubated with anti-phospho ERK p42/44 antibodies and visualized by ECL.
(Cohen, 1997)). Data fit well with findings by Strasser et al. (1995) who reported that apoptosis induced by g-irradiation was sensitive to Bcl-2/Bcl-XL treatment but insensitve to CrmA. In order to test the efficiency of CrmA additional experiments were performed. Transfected Jurkat Tag cells were treated with anti-Fas antibody. Transfection with CrmA resulted in a 86% reduction of apoptosis (data not shown). Bcl-2 is known to inhibit cell death induced by many stimuli whereby research regarding Bcl-2 has mainly focused on its role in directly regulating mitochondrial function. Bcl-2 knockout mice express a phenotype consistent with that of mice exposed to chronic oxidative stress. This has led to the hypothesis that Bcl-2-expressing cells have enhanced antioxidant capacities that suppress oxidative stress signals generated during the initiation phase of many apoptotic pathways (for a review see (Voehringer and Meyn, 2000)). Current results and earlier findings (for a review see (BaierBitterlich et al.,1997b; Wirleitner et al., 1998; Spˆttl et al., 2000)) imply the hypothesis that intracellular effects mediated by 7,8-dihydroneopterin may be intimately involved in regulating cellular redox pathways associated with mitochondrial function and apoptosis (Fig. 4). A potential effect of Bcl-2 on
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Fig. 4. Proposed signal transduction pathway of 7,8-dihydroneopterin-mediated apoptosis of Jurkat T-lymphocytes. Activated cell-mediated immunity is known to be accompanied by elevated concentrations of 7,8-dihydroneopterin which in high concentrations was found to interfere with the oxidant-antioxidant balance and may induce apoptosis. Current results showed that apoptosis of Jurkat T-lymphocytes was not affected by CrmA whereas it was significantly decreased upon cotransfection with Bcl-2 constructs. Results suggest that 7,8dihydroneopterin-induced apoptosis of T-lymphocytes is rather mediated by a Bcl-2-sensitive pathway and is independent of CrmAsensitive signal transduction pathways, e.g. Fas or TNF receptormediated apoptosis.
the production of reactive oxygen intermediates in 7,8-dihydroneopterin-induced apoptosis is an interesting possibility, since Kane et al. (1993) showed that Bcl-2 inhibited neural cell death by decreasing the net cellular generation of reactive oxygen species. Acknowledgements. This work was supported by the Austrian Ministry of Education, Science and Culture (GZ 70.024/2-Pr/4/97).
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European Journal of Cell Biology 81, 197 ± 202 (2002, April) ¥ ¹ Urban & Fischer Verlag ¥ Jena http://www.urbanfischer.de/journals/ejcb
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7,8-Dihydroneopterin induces apoptosis of Jurkat T-lymphocytes via a Bcl-2-sensitive pathway Christiane Enzingera, Barbara Wirleitnera, Christina Lutzb, G¸nther Bˆckc, Bettina Tomasellia, Gottfried Baierb, Dietmar Fuchsa, Gabriele Baier-Bitterlich1)a a
b c
Institute for Medical Chemistry and Biochemistry and Ludwig Boltzmann Institute for AIDS Research, Innsbruck/Austria Institute for Medical Biology and Human Genetics, Innsbruck/Austria Institute for Experimental Pathology, Innsbruck/Austria
Received August 20, 2001 Received in revised version December 19, 2001 Accepted December 20, 2001
Apoptosis ± pteridines ± oxidative stress ± CrmA ± Bcl-2 ± Jurkat T-lymphocytes ± 7,8-dihydroneopterin Activated cell-mediated immunity is known to be accompanied by elevated concentrations of 7,8-dihydroneopterin which in high concentrations was found to interfere with the oxidantantioxidant balance. In this study we investigated whether 7,8dihydroneopterin mediates apoptosis of Jurkat T-lymphocytes via a CrmA- or Bcl-2-sensitive pathway. Transient transfection assays with CrmA and Bcl-2 expression constructs showed that apoptosis was not affected by CrmAwhereas it was significantly decreased upon cotransfection with Bcl-2 constructs. Results suggest that 7,8-dihydroneopterin-induced apoptosis of Tlymphocytes is mediated by a Bcl-2-sensitive pathway.
Introduction Neopterin and 7,8-dihydroneopterin are synthesized in human macrophages upon induction with IFN-g (Huber et al., 1984; Bitterlich et al., 1988; Fuchs et al., 1988; Werner et al., 1989). In vivo, the production of neopterin closely correlates with IFN-g concentrations and the activation of cell-mediated immunity, e.g. in human immunodeficiency virus (HIV) infection, neopterin levels increase in parallel with progression of the disease (Fuchs et al., 1988, 1989). In vitro, the excretion of neopterin was found to correlate well with hydrogen peroxide levels (Nathan, 1986), and may therefore serve as a marker for oxidative stress in disease (Fuchs et al., 1997; Murr et al., 1999). By measurement of hydrogen peroxide-mediated luminol chemiluminescence, which allows indirect quantification of 1)
Professor Dr. Gabriele Baier-Bitterlich, Institute for Medical Chemistry and Biochemistry, Fritz Pregl Str. 3, A-6020 Innsbruck/ Austria, e-mail: [email protected], Fax: 43 512 507 2865.
reactive oxygen intermediates, a bimodal effect of 7,8-dihydroneopterin on hydrogen peroxide-mediated chemiluminescence was found. Lower concentrations scavenged, but at high doses (5 mM) 7,8-dihydroneopterin enhanced chemiluminescence (Weiss et al., 1993, Murr et al., 1994, 1996; BaierBitterlich et al., 1995; Reibnegger et al., 1995). This latter observation concedes with data in the literature (Blair and Pearson, 1973) that reduced pteridines react with molecular oxygen under formation of free radicals. At low concentrations it seems that 7,8-dihydroneopterin simply reacts as a chemical reductant destroying reactive oxygen intermediates. In further investigations, electron spin resonance measurements revealed that 7,8-dihydroneopterin in combination with the spin trap DMPO induces the production of DMPO-OH spin adducts, suggesting that 7,8-dihydroneopterin may give rise to the formation of OH radicals via generation of superoxide anion (Oettl et al., 1999; Wirleitner et al., 2001). In later experiments 7,8-dihydroneopterin was shown to function as an electron source needed to reduce dioxygen which led to the hypothesis that human monocytes/macrophages may strongly enhance their aggressive potential via the radical- generating capability of 7,8-dihydroneopterin in the presence of dioxygen (Reibnegger et al., 2001). Interestingly, neopterin and 7,8-dihydroneopterin were indeed found to interfere with redox-sensitive intracellular signaling pathways, and results implied a potential role of neopterin-derivatives in oxidative stress-mediated apoptosis (Fuchs et al., 1994a; Baier-Bitterlich et al., 1995, 1996a, b; Schobersberger et al., 1996; Wirleitner et al., 1998; Spˆttl et al., 2000). Upon incubation with high doses of 7,8dihydroneopterin, Jurkat T-lymphocytes revealed increased apoptosis by measurement of the intranuclear contents of fluorescing DNA by propidium iodide staining and FACS analysis (Baier-Bitterlich et al., 1996c). In order to study the potential modulatory effects of pteridines on mitochondrial function, isolated rat brain mitochondria were exposed to neopterin and 7,8-dihydroneopterin. Whereas exposure of
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mitochondria to either neopterin or 7,8-dihydroneopterin showed no effect on mitochondrial electron transport chain complex I, complex IV and citrate synthase activities, both compounds inhibited complex II/III in a dose-dependent manner (Wede and Fuchs, 1997). The effect of pharmacological inhibitors on 7,8-dihydroneopterin-mediated apoptosis was studied. While apoptosis was not inhibited by a broad range of pharmacological inhibitors such as actinomycin D, cycloheximide, cyclosporin A and various protein kinase inhibitors, inhibitors with antioxidant abilities such as pyrrolidinedithiocarbamate Nacetylcysteine (NAC), superoxid dismutase (SOD) and catalase (CAT) significantly blocked 7,8-dihydroneopterin-mediated apoptosis (Baier-Bitterlich et al., 1996c; Wirleitner et al., 1998). Reactive oxygen intermediates play an important role in cellular metabolic events such as signal transduction and regulation of gene expression (Meyer et al., 1994). As a consequence of oxidative stress a number of early response genes such as c-fos, c-myc and c-jun are transcriptionally activated (Crawford et al., 1988; Datta et al., 1992). Along this line it was observed that incubation of cells with neopterin or 7,8-dihydroneopterin induces c-fos gene expression (‹berall et al., 1994) and leads in conjunction with TNF-a to synergistic activation of NF-kB (Baier-Bitterlich et al., 1997a), which is also a well known oxidative stress responsive transcription factor (Schreck et al., 1991). Apoptosis induced by different stimuli in the same cell may be regulated by different pathways which are often CrmAsensitive or CrmA-resistant. While CrmA-sensitive pathways are frequently Bcl-2/Bcl-XL-resistant, the latter are Bcl-2/BclXL-sensitive (Cohen, 1997). Recently the oncogene Bcl-2 has attracted attention since its control function over apoptosis commitment in disease development and clinical response to therapies has been targeted for pharmacological intervention (for a review see (Voehringer and Meyn, 2000)). In this paper, we intended to investigate whether 7,8-dihydroneopterininduced apoptosis of T lymphocytes is mediated by Bcl-2- or CrmA-sensitive pathways. As a model system Jurkat Tlymphocytes were chosen which were transiently transfected with CrmA and Bcl-2 promoter-reporter gene constructs.
Material and methods Cell culture
CD4 Jurkat Tag cells (a kind gift from Dr. G. Crabtree, Stanford University, CA) which stably express the SV40-derived large T antigen were cultivated in RPMI-1640 medium (Biochrom, Berlin, FRG) complemented with 2 mM glutamine (Serva, FRG), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Biological Industries, Israel) and 10% heat-inactivated fetal calf serum (FCS, Biochrom, Berlin, FRG).
Reagents and plasmids 7,8-Dihydroneopterin was purchased from Schircks Lab. (Jona, Switzerland). The pEF-neo CrmA and Bcl-2 plasmids were a kind gift from Dr. R. Kofler (University of Innsbruck, Austria). The expression plasmid encoding the murine MHC class I protein H2-Kk was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Staurosporine was purchased from Sigma (Vienna, Austria).
Transfection of Jurkat T-cells to study the influence of Bcl-2 and Crm-A Jurkat Tag cells were double-transfected by electroporation with the indicated pEF-neo empty vector control, Bcl-2 or CrmA expression plasmids and the surface marker expression plasmid encoding the murine MHC class I H2-Kk, respectively, as follows. 24 hours after medium change JurkatTag cells were harvested by centrifugation and washed twice in medium without supplements at a concentration of 2 107 cells/400 ml. Aliquots of cell suspensions were transferred to Eppendorf vials and mixed with 20 mg plasmid DNA (neo, Bcl-2 or CrmA). To control for transfection efficiency, 10 mg of the control plasmid H2-Kk were subsequently added. Cells were transferred to the electroporation cuvettes, incubated for 10 min on ice and electroporated in a BTX-Electroporator T820 at 450 kV, 5 pulses, each 99 msec/3.0 kV. After electroporation, cells were incubated for 10 min at room temperature, resuspended in medium with supplements, seeded in culture plates and incubated for 48 hours. During this time cells (1 106) were stimulated with staurosporine (1 mM) or 7,8-dihydroneopterin (5 mM) for 6, 24 or 48 hours.
Detection of apoptotic cells Rates of apoptosis in transfected Jurkat cells were determined by flow cytometric evaluation of annexin-positive cells within the MHC I H2Kk-positive population 48 h after transfection. Cells were harvested and washed once in KR-buffer (Krebs Ringer-buffer; 145 mM NaCl, 5 mM KCl, 3 mM CaCl2, 10 mM glucose, 1.2 mM NaH2PO4, 1 mM MgSO4 and 20 mM Hepes), incubated in 200 ml KR-buffer containing phycoerythrin-labelled anti-mouse H2-Kk monoclonal antibody (1 ml/200 ml KRbuffer; Pharmingen, San Diego, CA, USA) to identify transfected cells and FITC-labelled annexin V (1 ml/200 ml KR-buffer; Alexis Biochemicals) for 30 min on ice. Finally, cells were washed, resuspended in 200 ml annexin binding buffer and analyzed by FACS-Scan. In parallel, cells were stained with Hoechst 33342 (Molecular probes, OR, USA) and analysed on an Olympus BX50 fluorescence microscope to characterize apoptotic nuclei.
Activation of p42/p44 mitogen-acivated protein kinase (MAP kinase) After electroporation cells were incubated for 10 min at room temperature, resuspended in medium with supplements, seeded in culture plates and incubated for 48 hours. During this time cells (1 106) were stimulated with 7,8-dihydroneopterin (5 mM) for 6 and 24 hours. To prepare cell lysates, cells were solubilised in 2 lysis buffer (100 mM Tris-HCl, pH 8.5, 2% NP-40, 10 mM EDTA, 10 mM NaF, 10 mM NaCl, 100 mM Na3VO4, 1 mg/ml aprotinin, 1 mg/ml leupeptin) and incubated for 20 min on ice. Following centrifugation (13 000g, 15 min, 4 8C), supernatants were analysed for protein content (Bradford protein assay, Bio-Rad, Vienna, Austria). 30 mg cell lysate protein were separated on an SDS polyacrylamide gel (12%) and transferred to nitrocellulose membranes (Bio-Rad). Immunoblotting was performed with anti-active MAP kinase antibodies (New England Biolabs, Inc., USA). Phosphorylated MAP kinase was detected with the ECL system (Amersham Pharmacia Biotech, UK). Values are representative of 2 (CrmA) to 3 experiments.
Results and discussion The induction of pteridine-mediated apoptosis of transfected Jurkat Tag cells was first analysed by Hoechst 33342 staining. A significant increase in apoptosis was induced by 7,8-dihydroneopterin (5 mM). Characteristics typical of apoptosis (e.g. chromatin condensation and formation of apoptotic bodies) were observed in transfected cells that were incubated with 7,8dihydroneopterin. Staurosporine was used as a positive control. (Fig. 1A ± D). Data correspond well with earlier findings in various other cell types (Baier-Bitterlich et al., 1995; Schobers-
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Fig. 1. 7,8-Dihydroneopterin-mediated apoptosis of transfected Jurkat T-lymphocytes. Jurkat Tag cells transfected with the control plasmid (PEF-neo) were incubated for 6 ± 24 hours with 7,8-dihydroneopterin (5 mM ) or staurosporine (1 mM) and stained with Hoechst 33342. Cells were analyzed on an Olympus BX50 fluorescence microscope (100 oil
immersion objective) to characterize apoptotic nuclei. (A) untreated control, (B) staurosporine (6 h), (C) 7,8-dihydroneopterin (6 h), (D) 7,8-dihydroneopterin (24 h). Micrographs are representative of 3 experiments.
berger et al., 1996; Spˆttl et al., 2000), e.g. neopterin and 7,8dihydroneopterin in conjunction with TNF-a were shown to intervene with the ability of U937 cells to maintain an appropriate oxidant-antioxidant balance. While concentrations of neopterin up to 1 mM and 7,8-dihydroneopterin up to 300 mM decreased TNF-induced apoptosis, higher doses of 7,8dihydroneopterin (1 mM) did not alter, and at a concentration of 5 mM even superinduced TNF-a-mediated apoptosis. Serum concentrations usually range from 5 nM in healthy individuals to 300 nM neopterin and to 1 mM 7,8-dihydroneopterin in severely ill patients (Fuchs et al., 1994b). Thus, the effective concentrations of 7,8-dihydroneopterin used in our cell culture approach appear to be unphysiological. Yet, in contrast to an only once added dose of stimulants in our experiments, a continuously high level of neopterin and 7,8dihydroneopterin persists in body fluids of patients due to continuous stimulation with IFN-g. Direct cell-to-cell contact may further facilitate the accumulation of even higher concentrations of 7,8-dihydroneopterin in the microenvironment of cells, and its effects may be further augmented in the concert of other inflammatory cytokines which are induced upon activation of cells with IFN-g (Billiau and Dijkmans, 1990) such as interleukin-1 and TNF-a.
Next the effect of CrmA and Bcl-2 on 7,8-dihydroneopterinmediated apoptosis was analysed. In control experiments cells were treated with staurosporine. As expected from recent data in the literature, Bcl-2 significantly decreased staurosporineinduced apoptosis after 24 hours while the inhibition by CrmA was less pronounced (e.g. (Yamashita et al., 2001; Mathiasen et al., 1999; Cohen, 1997)) (Fig. 2A). In contrast 7,8-dihydroneopterin-induced apoptosis was only inhibited in cells which were transfected with the Bcl-2 expression plasmid (Fig. 2B). Data fit well with investigations on the effect of 7,8-dihydroneopterin on MAP kinase (Fig. 3). The impact of 7,8-dihydroneopterin on the activity of the −survival ± associated× MAP kinase, in Jurkat cells transfected with CrmA or Bcl-2 expression plasmids was studied. MAP kinase activity in control cells and in CrmA-transfected cells had ceased after 24 hours but was still significantly elevated in cells expressing Bcl-2. This result again supports the previous observation of the impact of Bcl-2 expression on 7,8-dihydroneopterin-mediated apoptosis. The underlying molecular mechanism, however, remains to be elucidated. CrmA prevents apoptosis in a number of different systems, however its inhibiting capability is dependent on the stimulus to induce it. Apparently there are CrmA-sensitive and -resistant pathways present in the same cell type. CrmA is a
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Fig. 2. Effect of CrmA and Bcl-2 on (A) staurosporine- and (B) 7,8-dihydroneopterin-mediated apoptosis of Jurkat T-lymphocytes. Jurkat Tag cells (2 107 per experiment) were transfected with 20 mg plasmid DNA (pEF-neo, CrmA or Bcl-2) and 10 mg of the −transfection control− plasmid (H2-Kk -MHC I). After electroporation and a resting period cells were seeded in culture dishes and incubated for 48 hours. During this time cells (1 106) were stimulated with (A) staurosporine (1 mM) or (B) 7,8-dihydroneopterin (5 mM) for 6, 24 or 48 hours. Transfected cells were harvested, washed and incubated with phycoerythrin-labelled anti-mouse H2-Kk monoclonal antibody and FITC-labelled annexin V. Cells were analysed on a FACS SCAN. Values were expressed as fold of control (apoptosis (%): control pEF-neo 11.87 1.80; control Bcl-2 13.54 2.16; control CrmA 12.06 1.50) and represent the mean and SEM of 4 experiments. * p < 0.05.
poor inhibitor of CED-3, caspase-2, -3, -7, and -10, but an effective inhibitor of caspase-1, -4, -6 and -8 (see review by Cohen (1997)). Interestingly, many of the apoptotic pathways that are apparently sensitive to inhibition by CrmA are releatively resistant to Bcl-2/Bcl-XL, and vice versa (compare
Fig. 3. Activation of MAP kinase in transfected Jurkat T-cells. p42/44 MAP kinase activation was compared by immunoblotting in 7,8dihydroneopterin-treated Jurkat tag cells transfected with pEF-neo, CrmA or Bcl-2 expression constructs. Cells were pelleted and resuspended in lysis buffer. Cell lysates were cleared by centrifugation and equal amounts of protein were separated by SDS-PAGE. Blots were incubated with anti-phospho ERK p42/44 antibodies and visualized by ECL.
(Cohen, 1997)). Data fit well with findings by Strasser et al. (1995) who reported that apoptosis induced by g-irradiation was sensitive to Bcl-2/Bcl-XL treatment but insensitve to CrmA. In order to test the efficiency of CrmA additional experiments were performed. Transfected Jurkat Tag cells were treated with anti-Fas antibody. Transfection with CrmA resulted in a 86% reduction of apoptosis (data not shown). Bcl-2 is known to inhibit cell death induced by many stimuli whereby research regarding Bcl-2 has mainly focused on its role in directly regulating mitochondrial function. Bcl-2 knockout mice express a phenotype consistent with that of mice exposed to chronic oxidative stress. This has led to the hypothesis that Bcl-2-expressing cells have enhanced antioxidant capacities that suppress oxidative stress signals generated during the initiation phase of many apoptotic pathways (for a review see (Voehringer and Meyn, 2000)). Current results and earlier findings (for a review see (BaierBitterlich et al.,1997b; Wirleitner et al., 1998; Spˆttl et al., 2000)) imply the hypothesis that intracellular effects mediated by 7,8-dihydroneopterin may be intimately involved in regulating cellular redox pathways associated with mitochondrial function and apoptosis (Fig. 4). A potential effect of Bcl-2 on
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Fig. 4. Proposed signal transduction pathway of 7,8-dihydroneopterin-mediated apoptosis of Jurkat T-lymphocytes. Activated cell-mediated immunity is known to be accompanied by elevated concentrations of 7,8-dihydroneopterin which in high concentrations was found to interfere with the oxidant-antioxidant balance and may induce apoptosis. Current results showed that apoptosis of Jurkat T-lymphocytes was not affected by CrmA whereas it was significantly decreased upon cotransfection with Bcl-2 constructs. Results suggest that 7,8dihydroneopterin-induced apoptosis of T-lymphocytes is rather mediated by a Bcl-2-sensitive pathway and is independent of CrmAsensitive signal transduction pathways, e.g. Fas or TNF receptormediated apoptosis.
the production of reactive oxygen intermediates in 7,8-dihydroneopterin-induced apoptosis is an interesting possibility, since Kane et al. (1993) showed that Bcl-2 inhibited neural cell death by decreasing the net cellular generation of reactive oxygen species. Acknowledgements. This work was supported by the Austrian Ministry of Education, Science and Culture (GZ 70.024/2-Pr/4/97).
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