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Cyclosporin A inhibits activation of inducible nitric oxide synthase in C6 glioma cell line
Brain Research 816 Ž1999. 92–98
Research report
Cyclosporin A inhibits activation of inducible nitric oxide synthase in C6 glioma cell line Vladimir Trajkovic´ a , Vladimir Badovinac b , Vladimir Jankovic´ a , Marija Mostarica Stojkovic´ a
a, )
Institute of Microbiology and Immunology, School of Medicine, UniÕersity of Belgrade, Dr. Subotica ´ 1, 11000 Belgrade, YugoslaÕia b Institute for Biological Research, UniÕersity of Belgrade, 29 NoÕembra 142, 11000 Belgrade, YugoslaÕia Accepted 20 October 1998
Abstract The effects of immunosuppressant cyclosporin A ŽCsA. on nitric oxide ŽNO. production and inducible NO synthase ŽiNOS. mRNA expression in rat C6 glioma cell line were investigated. CsA applied simultaneously with iNOS activator IFN-g caused dose-dependent reduction of NO synthesis in confluent C6 cells, as determined by measuring accumulation of nitrite, an indicator of NO production, in 48 h culture supernatants. IFN-g-induced expression of iNOS, but not interferon regulatory factor-1 ŽIRF-1. mRNA was reduced in CsA-treated cells. The enzymatic activity of iNOS was not changed by CsA, since it failed to affect NO production in cells in which iNOS had already been induced with IFN-g and any further induction was blocked by protein synthesis inhibitor cycloheximide ŽCHX.. FK506 was not able to mimic inhibitory effect of CsA on NO production in C6 cells, suggesting calcineurin-independent mechanism of CsA action. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Cyclosporin A; FK506; Nitric oxide; iNOS; C6; Glioma; Rat
1. Introduction Nitric oxide ŽNO., a gaseous, short-lived free radical is an important signaling molecule involved in various physiological functions of mammalian organisms w34x, as well is in microbial and tumor cell killing w28x and target tissue damage in organ specific autoimmune diseases w27x. Recent studies also revealed immunoregulatory role of NO, as it was found to suppress T-cell and Th1 clones proliferation w1,41x as well as Th1 cytokine production w3,41x, thus downregulating Th1 response Žreviewed in Ref. w26x.. Excessive production of NO mediated by inducible NO synthase ŽiNOS. in cytokine activated astrocytes, microglia and infiltrating macrophages is associated with demyelinating diseases such as multiple sclerosis ŽMS. w4x and its animal model, experimental allergic encephalomyelitis ŽEAE. w24x. Inhibition of NO synthesis in EAE had opposing outcomes—attenuation w7,11,12x or aggravation w19,36x of disease, suggesting a complex, both neurotoxic and regulatory Žsuppression of Th1 response., neuroprotective ) C orresponding [email protected]
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role of NO. The immunosuppressive role of NO in EAE has been further confirmed by accentuated disease in mice with genetically disrupted iNOS gene w15x. Furthermore, since iNOS expressing astrocytes were not associated with inflammatory infiltrates in EAE, the proposed protective role could be associated with astrocyte-derived NO w42x. Therefore, exploring modulation of NO production in astroglial cells could contribute to better understanding of pathogenesis and finding new ways of treatment for autoimmune neurologic disorders. Cyclosporin A ŽCsA. is a powerful suppressor of the immune system, acting primarily as an inhibitor of T-cell activation, but has been reported to affect several other cell types w37x. Its actions are mediated through inhibition of Carcalmodulin-dependent phosphatase—calcineurin w37x. The most important clinical use of CsA is in prevention of allograft rejection w13x. Moreover, the therapeutic utility of this drug is being evaluated in a number of autoimmune diseases w13x. The effects of CsA therapy in EAE are dose dependent: high-dose CsA treatment downregulates the disease w6x, while low-dose treatment leads to paradoxical exacerbation and is accompanied by increased numbers IFN-g-producing cells w33,35x. Although CsA therapy showed some mild effects in MS, it cannot be recom-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 1 3 0 - 5
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mended at the present time because of the unfavorable ratio between beneficial and side effects w13x. It has been previously reported that CsA inhibits iNOS-dependent NO synthesis in murine macrophages w10,20x, rat renal mesangial cells w25x, and vascular smooth muscle cells w30x. Although the role of NO in CsA-sensitive demyelinating diseases of the central nervous system ŽEAE and MS. has been established, there are no reports concerning possible effects of CsA on NO production in glial cells. In this paper we demonstrate that CsA significantly reduced IFN-g-induced NO production and iNOS mRNA expression in astrocyte-derived C6 rat glioma cell line, not interfering with enzymatic activity of iNOS. This effect of CsA was probably independent of calcineurin inhibition, since FK506, a drug structurally unrelated to CsA but with shared mechanism of action w37x, failed to affect NO synthesis in C6 cells.
2. Materials and methods 2.1. Cells and reagents Rat glioma cell line C6 was obtained from European Collection of Animal Cell Cultures ŽSalisbury, UK.. Rat recombinant IFN-g was obtained from Holland Biotechnology ŽLeiden, The Netherlands.. CsA ŽSandimmun. was from Sandoz Pharma ŽBasel, Switzerland.. FK506 was purchased from Fujisawa Pharmaceutical ŽTokyo, Osaka.. Cycloheximide ŽCHX. was obtained from US Biochemical ŽCleveland, OH.. Sulfanilamide and naphthylethylenediamine dihydrochloride were from Sigma ŽSt. Louis, MO.. Moloney leukemia virus reverse transcriptase was obtained from Eurogentec ŽSeraing, Belgium.. Random primers were from Pharmacia ŽUppsala, Sweden.. 2.2. Cell cultures C6 cells were grown to confluence at 378C in 5% CO 2 humidified atmosphere in HEPES-buffered RPMI 1640 medium ŽFlow Lab, Irvine, UK. supplemented with 5% fetal calf serum ŽFlow., 2 mM L-glutamine, antibiotics and sodium pyruvate. After the conventional trypsinization procedure, cells Ž5 = 10 4rwell. were seeded in flat-bottomed 96-well plates ŽFlow. in 200 ml of culture medium and cultured until they attained confluence. Cells were then washed and incubated in 200 ml of fresh culture medium containing IFN-g Ž200 Urml. or IFN-g with CsA Ž0.0625–1 mM. or FK506 Ž0.625–10 mM.. Cells were cultured in triplicate wells for 48 h at 378C in humidified atmosphere with 5% CO 2 . To assess the influence of CsA on iNOS activity, after 24 h of cultivation with IFN-g Ž200 Urml., cells were washed and incubated for additional 48 h in 200 ml of fresh medium containing CHX Ž5 mgrml. or CHX and CsA Ž1 mM.. Cell viability was determined by Trypan blue exclusion.
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2.3. Nitrite measurement Nitrite accumulation, an indicator of NO production, was measured using the Griess reagent w22x. Briefly, 50 ml aliquots of culture supernatants from were mixed with an equal volume of Griess reagent Ža mixture at 1:1 of naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% H 3 PO4 . and incubated at room temperature for 10 min. The absorbance at 570 nm was measured in an automated Titertek Multiscan microplate reader ŽFlow.. The nitrite concentration Žin mM. was calculated from a NaNO 2 standard curve. 2.4. Determination of iNOS and interferon regulatory factor-1 (IRF-1) mRNA by RT–PCR Confluent C6 cells were incubated for 6 h ŽiNOS. or 3 h ŽIRF-1. with IFN-g or IFN-g and CsA Ž1 mM.. Total RNA was isolated by acid guanidinium thiocyanate–phenol–chloroform extraction w9x and reverse transcribed using Moloney leukemia virus reverse transcriptase and random primers. PCR amplification of cDNA with primers specific for iNOS or IRF-1 was carried out in the same tube with GAPDH primers and the products were visualized by electrophoresis through 2.5% agarose gel containing ethidium bromide. Number of cycles Ž35 for both iNOS and IRF-1, and 25 for GAPDH. ensuring nonsaturating PCR conditions was established in preliminary experiments. For the iNOS, primers were: sense, 5XA G A G A G A TC C G G TTC A C A -3 X ; antisense, 5 X CACAGAACTGAGGGTACA-3X and the PCR product was 377 bp. The IRF-1 primers were: sense, 5X-GACCAGAGCAGGAACAAG-3X ; antisense, 5X-TAACTTCCCTTCCTCATCC-3X and the PCR product was 414 bp. The primers for GAPDH were: sense, 5X-GAAGGGTGGGGCCAAAAG-3X ; antisense, 5X-GGATGCAGGGATGATGTTCT-3X and the PCR product was 295 bp. The PCR was performed in a Thermojet Eurogentec thermal cycle as follows: 30 s of denaturation at 958C, 30 s of annealing at 508 or 528 for iNOS and IRF-1, respectively, and 30 s of extension at 728C. 2.5. Statistical analysis The statistical significance of differences was analyzed using the Student’s t-test, and a P-value less than 0.05 was considered significant.
3. Results 3.1. CsA inhibits IFN-g-induced NO production in C6 glioma cells While C6 cells spontaneously produced barely detectable levels of nitrites Ž- 1 mM., treatment with IFN-g
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3.2. CsA does not affect iNOS actiÕity in C6 glioma cells To assess possible influence of CsA on iNOS catalytic activity, cells were pretreated for 24 h with IFN-g to induce iNOS, and CsA Ž1 mM. was added simultaneously with protein synthesis inhibitor CHX Ž5 mgrml. needed to prevent further induction of iNOS. Nitrite levels were then measured after an additional 48 h of cultivation. In these conditions CsA failed to affect NO production ŽFig. 2., indicating that this drug probably does not interfere with iNOS enzymatic activity in C6 cells, although protein synthesis-dependent events cannot be excluded. Similar inability of CsA to affect catalytic activity of iNOS was observed also by Hattori and Nakanishi w20x in J774 macrophage cell line. 3.3. CsA reduces iNOS, but not IRF-1 mRNA expression in IFN-g-treated C6 glioma cells
Fig. 1. Effects of CsA on IFN-g-induced NO production in C6 glioma cells. Confluent C6 cells were incubated with IFN-g Ž200 Urml. in the presence or absence of different concentrations of CsA Ž0.0625–1 mM.. Nitrite concentration in culture supernatants was determined after 48 h. Results are presented as mean"S.E.M. of five separate experiments Ž) p- 0.05, )) p- 0.01..
Ž200 Urml., a potent inducer of iNOS w28,34x caused a significant nitrite accumulation in C6 culture supernatants ŽFig. 1.. This effect was abrogated both with a protein synthesis inhibitor CHX Ž5 mgrml. and the aminoguanidine Ž1 mM., a selective inhibitor of iNOS w11x Žnot shown., indicating iNOS mediated L-arginine oxidation and consequent NO generation as a source of nitrite accumulation. Activation of iNOS transcription in C6 cells was further confirmed by elevated level of iNOS mRNA after IFN-g treatment ŽFig. 4.. This finding is concordant with previous results describing iNOS expression in LPSandror cytokine ŽIFN-g, TNF, IL-1.-stimulated rodent primary astrocytes and C6 glioma cells w14,18,21,40x. Treatm ent with CsA Ž 0.0625 – 1 m M . caused concentration-dependent decrease of nitrite levels in supernatants of IFN-g stimulated C6 cells ŽFig. 1.. CsA alone did not change very low basal NO production in C6 cells Žnot shown.. Cell viability was not affected by CsA, as assessed by trypan blue staining at the time of nitrite measurement, indicating specific interaction of CsA with intracellular events responsible for NO production in C6 glioma cells. However, at CsA concentrations higher than 5 mM significant loss of cell viability was observed Žnot shown., in concordance with previous finding describing apoptotic death in C6 cells treated with 50–500 mM CsA w31x.
To determine whether CsA affects steady-state iNOS mRNA levels in IFN-g-treated C6 cells, RT–PCR for iNOS mRNA was performed. Indeed, while IFN-g caused significant accumulation of iNOS mRNA in C6 cells, treatment with 1 mM CsA reduced this IFN-g-triggered iNOS mRNA expression, indicating that CsA might affect
Fig. 2. Effect of CsA on iNOS activity in C6 glioma cells. Confluent C6 cells were cultivated with IFN-g Ž200 Urml. for 24 h. Cells were washed and fresh medium containing 5 mgrml CHX Žcontrol., or CHX with CsA Ž1 mM. was added. Nitrite concentration in culture supernatants was determined after a further 48 h of cultivation. Results from a representative experiment, presented as percentage of control value, are means" S.E.M. of triplicate observations.
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iNOS activation at the transcriptional level, although interference with the mRNA stability cannot be excluded. Similar CsA-mediated reduction of iNOS mRNA, as a consequence of decreased transcription rate, was observed also in IL-1-treated rat renal mesangial cells w25x, as well as in rat vascular smooth muscle cells stimulated with IL-1 and TNF-a w30x. Since it has been shown recently that the transcription factor IRF-1 is essential for IFN-g-induced transcription of iNOS gene w23,29x, we assessed possible influence of CsA on IRF-1 mRNA expression in C6 cells ŽFig. 3.. However, while the level of IRF-1 mRNA was significantly elevated in IFN-g-stimulated cells compared to untreated ones, CsA did not have any discernible effect on this IFN-g-induced upregulation of IRF-1 mRNA. Basal expression of both iNOS and IRF-1 mRNA was unaffected by CsA alone Žnot shown.. Thus, the observed effect of CsA on iNOS mRNA expression was a consequence of CsA interference with some intracellular pathways not related to transcription of IRF-1. 3.4. FK506 does not mimic CsA-mediated inhibition of NO production in C6 glioma cells To test whether CsA-mediated inhibition of NO synthesis in C6 cells was due to its interference with calcineurindependent signaling pathways, we used FK506, a drug structurally unrelated to CsA, but also inhibiting calcineurin. However, in the same experimental conditions used to asses the effects of CsA, even higher concentra-
Fig. 4. Effects of FK506 on IFN-g-induced NO production in C6 glioma cells. Confluent C6 cells were incubated with IFN-g Ž200 Urml. in the presence or absence of different concentrations of FK506 Ž0.625–10 mM.. Nitrite concentration in culture supernatants was determined after 48 h. Results are presented as mean"S.E.M. of three separate experiments.
tions of FK506 Ž0.625–10 mM. failed to affect IFN-g-induced NO production in C6 cells ŽFig. 4., suggesting that inhibitory CsA action on NO synthesis in these cells might be unrelated to inhibition of calcineurin phosphatase activity. At concentrations higher than 20 mM, FK506 was toxic to C6 cells Žnot shown..
4. Discussion
Fig. 3. Effect of CsA on iNOS and IRF-1 mRNA expression in C6 glioma cells. Confluent C6 cells were incubated for 6 h with IFN-g Ž200 Urml., in the presence or absence of CsA Ž1 mM.. After isolation of RNA, RT–PCR using iNOS ŽA. or IRF-1 ŽB., together with GAPDH specific primers was carried out, and electrophoresis of PCR samples in agarose gel containing ethidium bromide was performed.
In this paper we showed that immunosuppressive drug CsA inhibits IFN-g-induced NO production in astrocytederived C6 glioma cell line. Our results suggest that CsA-mediated inhibition of NO synthesis was mediated through IRF-1-independent, as well as calcineurin-independent interference with iNOS induction in C6 cells, probably at the level of iNOS gene transcription. While similar effect of CsA on NO release have been described in rodent macrophages w10,20x, renal mesangial cells w25x, and vascular smooth muscle cells w30x, this is the first demonstration of CsA ability to influence NO synthesis and iNOS mRNA expression in cells of astroglial origin. Downregulation of iNOS activation by CsA, as proposed by Hattori and Nakanishi w20x for LPS treated macrophages, could be mediated through inhibition of
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secondary cytokine production, preventing them from contributing to iNOS induction in an autorparacrine fashion. Beside IFN-g, TNF-a and IL-1 are most potent inducers of iNOS in astrocytes and C6 cells w14,21x. However, it seems that IFN-g-induced NO production in astroglial cells is TNF-a-independent, considering inability of IFN-g to activate TNF-a synthesis in astrocytes w8x. It has been reported that even LPS, a potent inducer of macrophage TNF-a, could not activate its transcription in C6 glioma cells w17x. In addition, we observed that IFN-g did not enhance very low basal production of IL-1b in C6 cells, which remained unaffected also after CsA treatment Žunpublished observation.. Therefore, CsA probably does not suppress iNOS activation in IFN-g treated C6 cells by inhibiting secondary cytokine secretion, at least when TNF-a and IL-1 were concerned. On the other hand, CsA could have more direct influence on iNOS gene transcription. Binding of both IRF-1 and NF-kB to their consensus sequences in the iNOS promoter has been shown to be functionally important for the induction of rodent iNOS w23,29,43x. Although the IRF-1 is the principal mediator of IFN-g-triggered iNOS transcription in murine macrophages w23,29x, we were not able to demonstrate any effect of CsA on IFN-g-induced expression of mRNA for IRF-1. Therefore, CsA-mediated inhibition of iNOS activation in IFNg-treated C6 glioma cells probably was a consequence of CsA interference with some intracellular events unrelated to IRF-1 transcription. Recently, CsA has been found to inhibit NF-kB activation in T-cells and U937 monocytic cell line w16,38x. Furthermore, CsA-mediated inhibition of NF-kB activation is responsible for reduced iNOS transcription in IL-1-treated rat renal mesangial cells w25x. Although IFN-g is not considered as a typical NF-kB activator w2x, the abrogation of IFN-g-induced effects by interfering with constitutive activation of NF-kB has been described w39x. Nevertheless, the inhibition of NF-kB by CsA seems to be mediated through its well known interaction with calcineurin w16,38x, while inability of FK506 to mimic CsA inhibitory effect on NO synthesis in C6 glioma cells indicate calcineurin-independent CsA action. Thus, even if CsA-mediated interference with NF-kB is presumed, it is not likely to be a mechanism used by CsA to suppress iNOS activation in C6 cells, unless it was exerted independently of calcineurin inhibition. It is well known that calcineurin inhibition by immunophilin-binding drugs suppresses catalytic activity of constitutive NOS isoform through enhancement of its phosphorylation Žreviewed in Ref. w44x.. On the other hand, there are conflicting results concerning effects of immunophilin-binding immunosuppressants on catalytic activity of inducible isoform of NOS. While Conde et al. w10x observed direct suppressive effect of CsA and FK506 on iNOS enzymatic activity in murine peritoneal macrophages, Hattori and Nakanishi w20x, in concordance to our results on C6 cells, were not able to demonstrate it in J774 macrophage cell line. This discrepancy could be
easily due to the different cells used in these studies, since it has been shown that IL-13, another NO production inhibitor, affects different steps in NO synthesis activation in J774, compared to peritoneal macrophages w5x. The work of Hattori and Nakanishi also suggested that calcineurin inhibition might affect LPS-triggered induction of iNOS in J774 macrophages. However, since CsA was more potent than FK506 in inhibiting J774 macrophage NO synthesis, it is possible that suppressive action of CsA on iNOS induction in these cells could be partially independent of calcineurin inactivation. Moreover, inability of FK506 to mimic CsA-mediated suppression of iNOS activation in TNF-aq IL-1-stimulated vascular smooth muscle cells w30x, IL-1-treated mesangial cells w32x, as well as IFN-g-treated C6 glioma cell line, clearly suggest calcineurin-independent mechanism of NO synthesis inhibition by CsA. Therefore, it seems conceivable to presume that requirements for calcineurin phosphatase action in iNOS activation, as well as in its enzymatic activity might differ depending on cell type or iNOS stimuli used. The results from the present study could be also relevant for interpretation of results describing bimodal action of CsA in EAE. The proposed destructive and protective roles for NO in this disease, functioning as an effector molecule in demyelination as well as suppressing Th1 response, seem to correlate with macrophagermicroglial and astrocyte-derived NO, respectively w42x. Interestingly, CsA concentrations required for suppression of macrophage NO production w20x were approximately 20-fold higher than those causing similar inhibition of NO synthesis in C6 glioma cells. Thus, one could be tempted to assume that the selective inhibition of astrocyte NO synthesis might be partially responsible for the paradoxical exacerbation of EAE associated with low-dose CsA treatment. However, since the present study was performed with a rat astrocyte-like cell line, the implications of the results are limited. Although our preliminary results suggest similar effect of CsA on NO synthesis in rat primary astrocytes, further investigations on human astrocytes, as well as in vivo studies are needed.
Acknowledgements This work was supported by grants from Ministry of Science and Technology, Republic of Serbia, Yugoslavia.
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w43x Q.-W. Xie, Y. Kashiwabara, C. Nathan, Role of transcription factor NF-kBrRel in the induction of nitric oxide synthase, J. Biol. Chem. 269 Ž1994. 4705–4708. w44x J. Zhang, J.R. Steiner, Nitric oxide synthase, immunophilins and polyŽADP-ribose. synthetase: novel targets for the development of neuroprotective drugs, Neurol. Res. 269 Ž1995. 285–288.
Research report
Cyclosporin A inhibits activation of inducible nitric oxide synthase in C6 glioma cell line Vladimir Trajkovic´ a , Vladimir Badovinac b , Vladimir Jankovic´ a , Marija Mostarica Stojkovic´ a
a, )
Institute of Microbiology and Immunology, School of Medicine, UniÕersity of Belgrade, Dr. Subotica ´ 1, 11000 Belgrade, YugoslaÕia b Institute for Biological Research, UniÕersity of Belgrade, 29 NoÕembra 142, 11000 Belgrade, YugoslaÕia Accepted 20 October 1998
Abstract The effects of immunosuppressant cyclosporin A ŽCsA. on nitric oxide ŽNO. production and inducible NO synthase ŽiNOS. mRNA expression in rat C6 glioma cell line were investigated. CsA applied simultaneously with iNOS activator IFN-g caused dose-dependent reduction of NO synthesis in confluent C6 cells, as determined by measuring accumulation of nitrite, an indicator of NO production, in 48 h culture supernatants. IFN-g-induced expression of iNOS, but not interferon regulatory factor-1 ŽIRF-1. mRNA was reduced in CsA-treated cells. The enzymatic activity of iNOS was not changed by CsA, since it failed to affect NO production in cells in which iNOS had already been induced with IFN-g and any further induction was blocked by protein synthesis inhibitor cycloheximide ŽCHX.. FK506 was not able to mimic inhibitory effect of CsA on NO production in C6 cells, suggesting calcineurin-independent mechanism of CsA action. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Cyclosporin A; FK506; Nitric oxide; iNOS; C6; Glioma; Rat
1. Introduction Nitric oxide ŽNO., a gaseous, short-lived free radical is an important signaling molecule involved in various physiological functions of mammalian organisms w34x, as well is in microbial and tumor cell killing w28x and target tissue damage in organ specific autoimmune diseases w27x. Recent studies also revealed immunoregulatory role of NO, as it was found to suppress T-cell and Th1 clones proliferation w1,41x as well as Th1 cytokine production w3,41x, thus downregulating Th1 response Žreviewed in Ref. w26x.. Excessive production of NO mediated by inducible NO synthase ŽiNOS. in cytokine activated astrocytes, microglia and infiltrating macrophages is associated with demyelinating diseases such as multiple sclerosis ŽMS. w4x and its animal model, experimental allergic encephalomyelitis ŽEAE. w24x. Inhibition of NO synthesis in EAE had opposing outcomes—attenuation w7,11,12x or aggravation w19,36x of disease, suggesting a complex, both neurotoxic and regulatory Žsuppression of Th1 response., neuroprotective ) C orresponding [email protected]
author.
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role of NO. The immunosuppressive role of NO in EAE has been further confirmed by accentuated disease in mice with genetically disrupted iNOS gene w15x. Furthermore, since iNOS expressing astrocytes were not associated with inflammatory infiltrates in EAE, the proposed protective role could be associated with astrocyte-derived NO w42x. Therefore, exploring modulation of NO production in astroglial cells could contribute to better understanding of pathogenesis and finding new ways of treatment for autoimmune neurologic disorders. Cyclosporin A ŽCsA. is a powerful suppressor of the immune system, acting primarily as an inhibitor of T-cell activation, but has been reported to affect several other cell types w37x. Its actions are mediated through inhibition of Carcalmodulin-dependent phosphatase—calcineurin w37x. The most important clinical use of CsA is in prevention of allograft rejection w13x. Moreover, the therapeutic utility of this drug is being evaluated in a number of autoimmune diseases w13x. The effects of CsA therapy in EAE are dose dependent: high-dose CsA treatment downregulates the disease w6x, while low-dose treatment leads to paradoxical exacerbation and is accompanied by increased numbers IFN-g-producing cells w33,35x. Although CsA therapy showed some mild effects in MS, it cannot be recom-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 1 3 0 - 5
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mended at the present time because of the unfavorable ratio between beneficial and side effects w13x. It has been previously reported that CsA inhibits iNOS-dependent NO synthesis in murine macrophages w10,20x, rat renal mesangial cells w25x, and vascular smooth muscle cells w30x. Although the role of NO in CsA-sensitive demyelinating diseases of the central nervous system ŽEAE and MS. has been established, there are no reports concerning possible effects of CsA on NO production in glial cells. In this paper we demonstrate that CsA significantly reduced IFN-g-induced NO production and iNOS mRNA expression in astrocyte-derived C6 rat glioma cell line, not interfering with enzymatic activity of iNOS. This effect of CsA was probably independent of calcineurin inhibition, since FK506, a drug structurally unrelated to CsA but with shared mechanism of action w37x, failed to affect NO synthesis in C6 cells.
2. Materials and methods 2.1. Cells and reagents Rat glioma cell line C6 was obtained from European Collection of Animal Cell Cultures ŽSalisbury, UK.. Rat recombinant IFN-g was obtained from Holland Biotechnology ŽLeiden, The Netherlands.. CsA ŽSandimmun. was from Sandoz Pharma ŽBasel, Switzerland.. FK506 was purchased from Fujisawa Pharmaceutical ŽTokyo, Osaka.. Cycloheximide ŽCHX. was obtained from US Biochemical ŽCleveland, OH.. Sulfanilamide and naphthylethylenediamine dihydrochloride were from Sigma ŽSt. Louis, MO.. Moloney leukemia virus reverse transcriptase was obtained from Eurogentec ŽSeraing, Belgium.. Random primers were from Pharmacia ŽUppsala, Sweden.. 2.2. Cell cultures C6 cells were grown to confluence at 378C in 5% CO 2 humidified atmosphere in HEPES-buffered RPMI 1640 medium ŽFlow Lab, Irvine, UK. supplemented with 5% fetal calf serum ŽFlow., 2 mM L-glutamine, antibiotics and sodium pyruvate. After the conventional trypsinization procedure, cells Ž5 = 10 4rwell. were seeded in flat-bottomed 96-well plates ŽFlow. in 200 ml of culture medium and cultured until they attained confluence. Cells were then washed and incubated in 200 ml of fresh culture medium containing IFN-g Ž200 Urml. or IFN-g with CsA Ž0.0625–1 mM. or FK506 Ž0.625–10 mM.. Cells were cultured in triplicate wells for 48 h at 378C in humidified atmosphere with 5% CO 2 . To assess the influence of CsA on iNOS activity, after 24 h of cultivation with IFN-g Ž200 Urml., cells were washed and incubated for additional 48 h in 200 ml of fresh medium containing CHX Ž5 mgrml. or CHX and CsA Ž1 mM.. Cell viability was determined by Trypan blue exclusion.
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2.3. Nitrite measurement Nitrite accumulation, an indicator of NO production, was measured using the Griess reagent w22x. Briefly, 50 ml aliquots of culture supernatants from were mixed with an equal volume of Griess reagent Ža mixture at 1:1 of naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% H 3 PO4 . and incubated at room temperature for 10 min. The absorbance at 570 nm was measured in an automated Titertek Multiscan microplate reader ŽFlow.. The nitrite concentration Žin mM. was calculated from a NaNO 2 standard curve. 2.4. Determination of iNOS and interferon regulatory factor-1 (IRF-1) mRNA by RT–PCR Confluent C6 cells were incubated for 6 h ŽiNOS. or 3 h ŽIRF-1. with IFN-g or IFN-g and CsA Ž1 mM.. Total RNA was isolated by acid guanidinium thiocyanate–phenol–chloroform extraction w9x and reverse transcribed using Moloney leukemia virus reverse transcriptase and random primers. PCR amplification of cDNA with primers specific for iNOS or IRF-1 was carried out in the same tube with GAPDH primers and the products were visualized by electrophoresis through 2.5% agarose gel containing ethidium bromide. Number of cycles Ž35 for both iNOS and IRF-1, and 25 for GAPDH. ensuring nonsaturating PCR conditions was established in preliminary experiments. For the iNOS, primers were: sense, 5XA G A G A G A TC C G G TTC A C A -3 X ; antisense, 5 X CACAGAACTGAGGGTACA-3X and the PCR product was 377 bp. The IRF-1 primers were: sense, 5X-GACCAGAGCAGGAACAAG-3X ; antisense, 5X-TAACTTCCCTTCCTCATCC-3X and the PCR product was 414 bp. The primers for GAPDH were: sense, 5X-GAAGGGTGGGGCCAAAAG-3X ; antisense, 5X-GGATGCAGGGATGATGTTCT-3X and the PCR product was 295 bp. The PCR was performed in a Thermojet Eurogentec thermal cycle as follows: 30 s of denaturation at 958C, 30 s of annealing at 508 or 528 for iNOS and IRF-1, respectively, and 30 s of extension at 728C. 2.5. Statistical analysis The statistical significance of differences was analyzed using the Student’s t-test, and a P-value less than 0.05 was considered significant.
3. Results 3.1. CsA inhibits IFN-g-induced NO production in C6 glioma cells While C6 cells spontaneously produced barely detectable levels of nitrites Ž- 1 mM., treatment with IFN-g
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3.2. CsA does not affect iNOS actiÕity in C6 glioma cells To assess possible influence of CsA on iNOS catalytic activity, cells were pretreated for 24 h with IFN-g to induce iNOS, and CsA Ž1 mM. was added simultaneously with protein synthesis inhibitor CHX Ž5 mgrml. needed to prevent further induction of iNOS. Nitrite levels were then measured after an additional 48 h of cultivation. In these conditions CsA failed to affect NO production ŽFig. 2., indicating that this drug probably does not interfere with iNOS enzymatic activity in C6 cells, although protein synthesis-dependent events cannot be excluded. Similar inability of CsA to affect catalytic activity of iNOS was observed also by Hattori and Nakanishi w20x in J774 macrophage cell line. 3.3. CsA reduces iNOS, but not IRF-1 mRNA expression in IFN-g-treated C6 glioma cells
Fig. 1. Effects of CsA on IFN-g-induced NO production in C6 glioma cells. Confluent C6 cells were incubated with IFN-g Ž200 Urml. in the presence or absence of different concentrations of CsA Ž0.0625–1 mM.. Nitrite concentration in culture supernatants was determined after 48 h. Results are presented as mean"S.E.M. of five separate experiments Ž) p- 0.05, )) p- 0.01..
Ž200 Urml., a potent inducer of iNOS w28,34x caused a significant nitrite accumulation in C6 culture supernatants ŽFig. 1.. This effect was abrogated both with a protein synthesis inhibitor CHX Ž5 mgrml. and the aminoguanidine Ž1 mM., a selective inhibitor of iNOS w11x Žnot shown., indicating iNOS mediated L-arginine oxidation and consequent NO generation as a source of nitrite accumulation. Activation of iNOS transcription in C6 cells was further confirmed by elevated level of iNOS mRNA after IFN-g treatment ŽFig. 4.. This finding is concordant with previous results describing iNOS expression in LPSandror cytokine ŽIFN-g, TNF, IL-1.-stimulated rodent primary astrocytes and C6 glioma cells w14,18,21,40x. Treatm ent with CsA Ž 0.0625 – 1 m M . caused concentration-dependent decrease of nitrite levels in supernatants of IFN-g stimulated C6 cells ŽFig. 1.. CsA alone did not change very low basal NO production in C6 cells Žnot shown.. Cell viability was not affected by CsA, as assessed by trypan blue staining at the time of nitrite measurement, indicating specific interaction of CsA with intracellular events responsible for NO production in C6 glioma cells. However, at CsA concentrations higher than 5 mM significant loss of cell viability was observed Žnot shown., in concordance with previous finding describing apoptotic death in C6 cells treated with 50–500 mM CsA w31x.
To determine whether CsA affects steady-state iNOS mRNA levels in IFN-g-treated C6 cells, RT–PCR for iNOS mRNA was performed. Indeed, while IFN-g caused significant accumulation of iNOS mRNA in C6 cells, treatment with 1 mM CsA reduced this IFN-g-triggered iNOS mRNA expression, indicating that CsA might affect
Fig. 2. Effect of CsA on iNOS activity in C6 glioma cells. Confluent C6 cells were cultivated with IFN-g Ž200 Urml. for 24 h. Cells were washed and fresh medium containing 5 mgrml CHX Žcontrol., or CHX with CsA Ž1 mM. was added. Nitrite concentration in culture supernatants was determined after a further 48 h of cultivation. Results from a representative experiment, presented as percentage of control value, are means" S.E.M. of triplicate observations.
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iNOS activation at the transcriptional level, although interference with the mRNA stability cannot be excluded. Similar CsA-mediated reduction of iNOS mRNA, as a consequence of decreased transcription rate, was observed also in IL-1-treated rat renal mesangial cells w25x, as well as in rat vascular smooth muscle cells stimulated with IL-1 and TNF-a w30x. Since it has been shown recently that the transcription factor IRF-1 is essential for IFN-g-induced transcription of iNOS gene w23,29x, we assessed possible influence of CsA on IRF-1 mRNA expression in C6 cells ŽFig. 3.. However, while the level of IRF-1 mRNA was significantly elevated in IFN-g-stimulated cells compared to untreated ones, CsA did not have any discernible effect on this IFN-g-induced upregulation of IRF-1 mRNA. Basal expression of both iNOS and IRF-1 mRNA was unaffected by CsA alone Žnot shown.. Thus, the observed effect of CsA on iNOS mRNA expression was a consequence of CsA interference with some intracellular pathways not related to transcription of IRF-1. 3.4. FK506 does not mimic CsA-mediated inhibition of NO production in C6 glioma cells To test whether CsA-mediated inhibition of NO synthesis in C6 cells was due to its interference with calcineurindependent signaling pathways, we used FK506, a drug structurally unrelated to CsA, but also inhibiting calcineurin. However, in the same experimental conditions used to asses the effects of CsA, even higher concentra-
Fig. 4. Effects of FK506 on IFN-g-induced NO production in C6 glioma cells. Confluent C6 cells were incubated with IFN-g Ž200 Urml. in the presence or absence of different concentrations of FK506 Ž0.625–10 mM.. Nitrite concentration in culture supernatants was determined after 48 h. Results are presented as mean"S.E.M. of three separate experiments.
tions of FK506 Ž0.625–10 mM. failed to affect IFN-g-induced NO production in C6 cells ŽFig. 4., suggesting that inhibitory CsA action on NO synthesis in these cells might be unrelated to inhibition of calcineurin phosphatase activity. At concentrations higher than 20 mM, FK506 was toxic to C6 cells Žnot shown..
4. Discussion
Fig. 3. Effect of CsA on iNOS and IRF-1 mRNA expression in C6 glioma cells. Confluent C6 cells were incubated for 6 h with IFN-g Ž200 Urml., in the presence or absence of CsA Ž1 mM.. After isolation of RNA, RT–PCR using iNOS ŽA. or IRF-1 ŽB., together with GAPDH specific primers was carried out, and electrophoresis of PCR samples in agarose gel containing ethidium bromide was performed.
In this paper we showed that immunosuppressive drug CsA inhibits IFN-g-induced NO production in astrocytederived C6 glioma cell line. Our results suggest that CsA-mediated inhibition of NO synthesis was mediated through IRF-1-independent, as well as calcineurin-independent interference with iNOS induction in C6 cells, probably at the level of iNOS gene transcription. While similar effect of CsA on NO release have been described in rodent macrophages w10,20x, renal mesangial cells w25x, and vascular smooth muscle cells w30x, this is the first demonstration of CsA ability to influence NO synthesis and iNOS mRNA expression in cells of astroglial origin. Downregulation of iNOS activation by CsA, as proposed by Hattori and Nakanishi w20x for LPS treated macrophages, could be mediated through inhibition of
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secondary cytokine production, preventing them from contributing to iNOS induction in an autorparacrine fashion. Beside IFN-g, TNF-a and IL-1 are most potent inducers of iNOS in astrocytes and C6 cells w14,21x. However, it seems that IFN-g-induced NO production in astroglial cells is TNF-a-independent, considering inability of IFN-g to activate TNF-a synthesis in astrocytes w8x. It has been reported that even LPS, a potent inducer of macrophage TNF-a, could not activate its transcription in C6 glioma cells w17x. In addition, we observed that IFN-g did not enhance very low basal production of IL-1b in C6 cells, which remained unaffected also after CsA treatment Žunpublished observation.. Therefore, CsA probably does not suppress iNOS activation in IFN-g treated C6 cells by inhibiting secondary cytokine secretion, at least when TNF-a and IL-1 were concerned. On the other hand, CsA could have more direct influence on iNOS gene transcription. Binding of both IRF-1 and NF-kB to their consensus sequences in the iNOS promoter has been shown to be functionally important for the induction of rodent iNOS w23,29,43x. Although the IRF-1 is the principal mediator of IFN-g-triggered iNOS transcription in murine macrophages w23,29x, we were not able to demonstrate any effect of CsA on IFN-g-induced expression of mRNA for IRF-1. Therefore, CsA-mediated inhibition of iNOS activation in IFNg-treated C6 glioma cells probably was a consequence of CsA interference with some intracellular events unrelated to IRF-1 transcription. Recently, CsA has been found to inhibit NF-kB activation in T-cells and U937 monocytic cell line w16,38x. Furthermore, CsA-mediated inhibition of NF-kB activation is responsible for reduced iNOS transcription in IL-1-treated rat renal mesangial cells w25x. Although IFN-g is not considered as a typical NF-kB activator w2x, the abrogation of IFN-g-induced effects by interfering with constitutive activation of NF-kB has been described w39x. Nevertheless, the inhibition of NF-kB by CsA seems to be mediated through its well known interaction with calcineurin w16,38x, while inability of FK506 to mimic CsA inhibitory effect on NO synthesis in C6 glioma cells indicate calcineurin-independent CsA action. Thus, even if CsA-mediated interference with NF-kB is presumed, it is not likely to be a mechanism used by CsA to suppress iNOS activation in C6 cells, unless it was exerted independently of calcineurin inhibition. It is well known that calcineurin inhibition by immunophilin-binding drugs suppresses catalytic activity of constitutive NOS isoform through enhancement of its phosphorylation Žreviewed in Ref. w44x.. On the other hand, there are conflicting results concerning effects of immunophilin-binding immunosuppressants on catalytic activity of inducible isoform of NOS. While Conde et al. w10x observed direct suppressive effect of CsA and FK506 on iNOS enzymatic activity in murine peritoneal macrophages, Hattori and Nakanishi w20x, in concordance to our results on C6 cells, were not able to demonstrate it in J774 macrophage cell line. This discrepancy could be
easily due to the different cells used in these studies, since it has been shown that IL-13, another NO production inhibitor, affects different steps in NO synthesis activation in J774, compared to peritoneal macrophages w5x. The work of Hattori and Nakanishi also suggested that calcineurin inhibition might affect LPS-triggered induction of iNOS in J774 macrophages. However, since CsA was more potent than FK506 in inhibiting J774 macrophage NO synthesis, it is possible that suppressive action of CsA on iNOS induction in these cells could be partially independent of calcineurin inactivation. Moreover, inability of FK506 to mimic CsA-mediated suppression of iNOS activation in TNF-aq IL-1-stimulated vascular smooth muscle cells w30x, IL-1-treated mesangial cells w32x, as well as IFN-g-treated C6 glioma cell line, clearly suggest calcineurin-independent mechanism of NO synthesis inhibition by CsA. Therefore, it seems conceivable to presume that requirements for calcineurin phosphatase action in iNOS activation, as well as in its enzymatic activity might differ depending on cell type or iNOS stimuli used. The results from the present study could be also relevant for interpretation of results describing bimodal action of CsA in EAE. The proposed destructive and protective roles for NO in this disease, functioning as an effector molecule in demyelination as well as suppressing Th1 response, seem to correlate with macrophagermicroglial and astrocyte-derived NO, respectively w42x. Interestingly, CsA concentrations required for suppression of macrophage NO production w20x were approximately 20-fold higher than those causing similar inhibition of NO synthesis in C6 glioma cells. Thus, one could be tempted to assume that the selective inhibition of astrocyte NO synthesis might be partially responsible for the paradoxical exacerbation of EAE associated with low-dose CsA treatment. However, since the present study was performed with a rat astrocyte-like cell line, the implications of the results are limited. Although our preliminary results suggest similar effect of CsA on NO synthesis in rat primary astrocytes, further investigations on human astrocytes, as well as in vivo studies are needed.
Acknowledgements This work was supported by grants from Ministry of Science and Technology, Republic of Serbia, Yugoslavia.
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