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Negatively charged phospholipids suppress IFN-γ production in T cells
BBRC Biochemical and Biophysical Research Communications 338 (2005) 1719–1725 www.elsevier.com/locate/ybbrc
Negatively charged phospholipids suppress IFN-c production in T cells Satoshi Yotsumoto a, Terutaka Kakiuchi b, Yukihiko Aramaki a,* a
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan b Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143-8540, Japan Received 25 October 2005 Available online 7 November 2005
Abstract The effect of phospholipids on IFN-c production in mouse T cells was investigated. Phosphatidylserine (PS), which has a negatively charged head group, completely inhibited IFN-c production in splenic naı¨ve T cells and antigen-dependent IFN-c production in Th1 clone 42-6A cells, whereas other phospholipids, which have neutrally charged head group, had no effect. The structural requirements for IFN-c inhibitory effects by PS were investigated, and dimyristoyl-PS (C14: 0) and dipalmitoyl-PS (C16: 0) had no effect on IFN-c production, and interestingly, distearoyl-PS (18: 0) increased IFN-c production. Dioleoyl-PS (C18: 1), dilinoleoyl-PS (C18: 2), and oleoyl-lyso-PS (C18: 1) completely inhibited IFN-c production. To clarify this mechanism, we focused on the stability of IFN-c mRNA, and the treatment of splenic naı¨ve T cells with PS brought about 40% reductions in IFN-c mRNA expression in the presence of actinomycin D. Collectively, IFN-c inhibitory effects by PS are highly dependent on the molecular structure of PS and involve the decreasing of the stability of IFN-c mRNA. 2005 Elsevier Inc. All rights reserved. Keywords: Phospholipids; T cell; IFN-c; Phosphatidylserine; Molecular structure
Phospholipids in the mammalian plasma membrane consist of various phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine (PS), phosphatidic acid, and phosphatidylinositol. Phospholipid molecules have a polar head group and two hydrophobic hydrocarbon tails. The tails are fatty acids and can differ in length. The tails usually have one or more cis-double bonds (unsaturated bonds). PS is a major anionic phospholipid component of mammalian cell membranes. Recent studies have shown that PS is an essential cofactor for the activation of protein kinase C [1–3] and blood coagulation [4,5]. PS has also been shown to regulate the activities of various enzymes, such as Na+/K+-ATPase [6], diacylglycerol kinase [7], B-Raf protein kinase [8], dynamin GTPase [9], and glutamate [10]. IFN-c, mainly produced by T cells and NK cells, is a multifunctional cytokine known to be a regulator of numerous immunological functions. Especially, IFN-c plays an impor*
Corresponding author. Fax: +81 426 76 3183. E-mail address: [email protected] (Y. Aramaki).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.179
tant role in the elimination of tumor cells by cytotoxic T lymphocytes. It is well known that IFN-c production is dramatically reduced in T cells from mice bearing large mammary tumors [11]. A recent report suggested that various phospholipids were produced by a D1-DMBA-3 mammary tumor and may contribute to the down-regulation of IFN-c production in T cells from mice bearing large D1DMBA-3 mammary tumors [12,13]. However, it is not clear that how the molecular structure of phospholipids is important for the IFN-c inhibitory effect. In this paper, we investigated the effect of the molecular structure of phospholipids on IFN-c production in mouse T cells. The findings suggested that negatively charged phospholipids inhibited IFN-c production and that the inhibitory effect is highly dependent on the molecular structure of negatively charged phospholipids. Materials and methods Materials. Phosphatidylcholine (PC) was purchased from Nippon Oil and Fat (Tokyo, Japan). Ovalbumin (OVA), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidyl-
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serine (PS), and Quantum Red-labeled anti-Thy1.2 monoclonal antibody were purchased from Sigma Chemical (St. Louis, MO). Anti-mouse CD3 e monoclonal antibody and recombinant mouse IL-2 were obtained from Genzyme (Cambridge, MA). Dimyristoyl-PS (C14: 0), dipalmitoyl-PS (C16: 0), distearoyl-PS (18: 0), dioleoyl-PS (C18: 1), dilinoleoyl-PS (C18: 2), and oleoyl-lyso-PS (C18: 1 were purchased from Avanti Polar Lipids (Alabaster, AL). Animals. BALB/c mice were purchased from Japan SLC (Shizuoka, Japan). All mice were used at 6–10 weeks of age. Animal use and relevant experimental procedures were approved by the Tokyo University of Pharmacy and Life Science Committee on the care and use of laboratory animals. Preparation of splenic naı¨ve T cells. Splenic naı¨ve T cells were purified by passing spleen cells through nylon wool columns twice. After washing twice with PBS, the cells were suspended in RPMI1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.28% NaHCO3, 10 mM Hepes, 5 · 105 M of 2-mercaptoethanol, 100 U/ml penicillin, and 100 lg/ml streptomycin in a humidified 5% CO2 at 37 C. Cell purity was ascertained by flow cytometric analysis using FACScalibur (Becton– Dickinson, Mountain View, CA). Routinely, the naı¨ve T cell preparations had a purity of 90–95%. T cell clones. Ovalbumin (OVA)-specific Th1 clones, 42-6A cells established from BALB/c mice [14] were maintained in RPMI1640 medium supplemented with 10 U/ml murine recombinant IL-2. Preparation of splenic adherent cells. Spleen cells from BALB/c mice were incubated in 100-mm plastic dishes (Iwaki glass, Chiba, Japan) for 90 min at 37 C in RPMI1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.28% NaHCO3, 10 mM Hepes, 50 lM of 2mercaptoethanol, 100 U/ml penicillin, and 100 lg/ml streptomycin. Nonadherent cells were removed by washing the dishes five times with icecold PBS. Splenic adherent cells (SAC) were recovered by 0.5% lidocaine– PBS (Astrazeneca, Osaka, Japan). SAC were washed twice and resuspended in RPMI1640 medium. SAC used as antigen presenting cells for OVA-specific Th1 clones, 42-6A cells. Preparation of phospholipid suspension. Phospholipids in chloroform solution were dried in a test tube under N2 gas and desiccated under vacuum for at least 2 h. The dry lipid films were hydrated by the addition of 1 ml of 10 mM Tris-buffer saline (pH 7.2) and the phospholipid suspension was prepared. The suspensions were then sterilized by passing through a membrane filter (0.45 lm; Iwaki, Tokyo, Japan). Determination of murine IFN-c. The levels of IFN-c in culture supernatants were routinely assayed by sandwich ELISA using pairs of purified
A
capture and biotinylated detection monoclonal antibodies recognizing murine IFN-c, according to the manufacturerÕs protocols. Association of phospholipids. Splenic T cells were incubated with NBDlabeled phospholipids at 37 C for 1 h. The cells were washed with ice-cold PBS and stained with Quantum Red-labeled anti-Thy1.2 mAb at 4 C for 30 min. After washing, the cells were fixed with 4% paraformaldehyde– PBS, and the association and localization of NBD-phospholipids were examined using flow cytometry or confocal microscopy, respectively. RT-PCR analysis. IFN-c mRNA expressions were determined using RT-PCR. Total RNA was isolated from splenic naı¨ve T cells with Isogen solution (Nippon Gene, Toyama, Japan). cDNAs were synthesized using SuperScript. cDNA were then amplified with specific primers for IFN-c. Each primer was designed based on the mouse sequences (IFN-c—forward, 5 0 -GACTTCAAAGATCTGAGG-3; IFN-c—reverse, 5 0 -AACGCT ACACACTGCATCTTGG-3 0 ; b-actin—forward, 5 0 -GCACCACACCTT CTACAATGA-G-3 0 ; b-actin—reverse, 5 0 -TTGGCATAGAGGTCTTTA CGGA-3 0 ). PCR was performed for 26 cycles of denaturation at 94 C for 60 s, annealing at 55 C for 60 s, and extension at 72 C for 60 s. The amplified products were analyzed on 2% agarose gels containing 0.1 lg/ml ethidium bromide. The band intensity was analyzed with NIH Image.
Results and discussion IFN-c production in splenic T cells is inhibited by negatively charged phospholipids We investigated the effects of various natural phospholipids on IFN-c production in mouse splenic T cells. As shown in Fig. 1A, IFN-c production elicited by the mitogen, 5 lg/ml concanavalin A (Con A), was inhibited when splenic T cells were treated with PS and PA (each 50 lg/ ml), which have a negatively polar head group. No inhibitory effects were observed in PC, PI, and PE (50 lg/ml), which have a neutral head group. PS and PA showed dose-dependent inhibitory effects on IFN-c production, and complete inhibition was observed in PS at 50 lg/ml and in PA at 200 lg/ml (Fig. 1B). A higher concentration of PA was required to inhibit IFN-c production compared
B
Fig. 1. Effects of various phospholipids on IFN-c production. (A) Splenic naı¨ve T cells were incubated for 18 h with Con A (2.5 lg/ml) in the presence of PC, PI, PE, PS or PA (50 lg/ml). (B) Effects of PS or PA doses on IFN-c production. Splenic naı¨ve T cells were incubated for 18 h with Con A (2.5 lg/ml) in the presence of PS (0–100 lg/ml) or PA (0–200 lg/ml). The supernatants of splenic T cell cultures were collected and IFN-c levels were measured as described under Materials and methods. The values are means ± SD of triplicate cultures from three independent experiments.
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with that of PS. The viability of splenic T cells after PS treatment was estimated with a trypan blue dye exclusion test, and no changes in viability were observed at the concentrations used (data not shown). These results suggest that phospholipids, which have a negatively charged polar head group, inhibit IFN-c production in splenic T cells without a reduction in cell viability. Effect of PS on IFN-c production induced by various stimulations The effects of PS on IFN-c production in splenic T cells elicited by various stimulants were investigated. As shown in Fig. 2, IFN-c production in splenic T cells elicited by anti-CD3e mAb (10 lg/ml), or 50 ng/ml phorbol ester plus ionomycin (500 ng/ml), was also completely inhibited by the treatment with PS (50 lg/ml). PC did not affect the IFN-c production. These results indicate that PS has the ability to inhibit IFN-c production in splenic T cells irrespective of the presence of stimulants. Effect of PS on antigen-specific IFN-c production in Th1 clone cells Naı¨ve CD4+ T cells differentiate into T helper 1 (Th1) or Th2 cells depending upon the cytokine environment. Th1 cells produce IL-2 and IFN-c, whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13 [15]. It is well known that IFN-c production by Th1 cells is induced when the cells are cocultured with antigen presenting cells (APC), such as macrophage/dendritic cells, in the presence of antigens (designated as antigen-dependent IFN-c production) [16,17]. Next, we investigated the effect of PS on antigendependent IFN-c production using OVA-specific Th1 clone 42-6A cells. As shown in Fig. 3, antigen-dependent IFN-c production in 42-6A cells, which were cocultured with SAC in the presence of 2 mg/ml OVA, was completely
Fig. 3. Effect of PS on antigen-dependent IFN-c production. The coculture of 42-6A cells with SAC was incubated for 18 h with OVA (2 mg/ml) in the presence of PC or PS. The supernatants of 42-6A cell cultures were collected and IFN-c levels were measured, as described under Materials and methods. The values are means ± SD of triplicate cultures from three independent experiments.
inhibited by PS in a dose-dependent manner, but not PC. Xiaofang et al. previously reported that PS effectively down-regulated the IFN-c production in naı¨ve CD4+ T cells from the spleen in vitro [13]. In this study, we indicate that PS inhibits antigen-dependent IFN-c production from differentiated T cells, such as CD4+ Th1 cells. Studies are now underway to clarify whether IFN-c production in CD8+ cytotoxic T lymphocytes was inhibited by PS. Association and localization of PS To clarify whether the inhibition of IFN-c production by phospholipids resulted from differences in the amount of phospholipids taken up, the association (sum of bind-
Fig. 2. Effects of PS on IFN-c production in splenic naı¨ve T cells with various stimulations. Splenic naı¨ve T cells were incubated for 18 h with anti-CD3e mAb (10 lg/ml), or PMA (50 ng/ml) plus ionomycin (500 ng/ml) in the presence of PC or PS (0–50 lg/ml). The supernatants of splenic T cell cultures were collected and the IFN-c levels were measured as described under Materials and methods. The values are means ± SD of triplicate cultures from three independent experiments.
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ing and uptake) of phospholipids was studied using NBDlabeled phospholipids. NBD is a fluorescence probe and the association of phospholipids can easily be evaluated using flow cytometry and confocal laser scanning microscopy. As shown in Fig. 4A, the fluorescence of cells incubated with NBD-labeled PS (NBD-PS) was observed, but that of NBD-PC was not. Furthermore, NBD-PS was not colocalized with Quantum Red-labeled monoclonal antibody against Thy1.2, a cell surface marker of T cells (Fig. 4B). These results suggest that the difference in the amount of phospholipids associated with T cells was involved in the induction of IFN-c inhibitory effects and PS was not localized to the cell surface. A previous report [18] suggested that exogenous PS is mainly transported to the Golgi apparatus by three consecutive processes in CHO-K1 cells. They are: (a) the insertion of PS into the outer leaflet of the plasma membrane, (b) translocation of PS from the outer to the inner leaflet of the plasma membrane, and (c) transport of PS from the inner leaflet
of the plasma membrane to the Golgi apparatus via a nonvesicular pathway. It is likely that PS, localized in inner leaflet of the plasma membrane or Golgi apparatus, affects T cell function. Structure–activity relationship of IFN-c inhibitory effects by PS Stearoyl-PS (18: 0) and oleoyl-PS (C18: 1) are major components of PS produced by human D1-DMBA-3 tumor cells [19]. However, it is not clear that how the molecular structure of PS is important for IFN-c inhibitory effects by PS. We thus next examined the structural requirements for IFN-c inhibitory effects by PS. First, the effects of the length of hydrophobic hydrocarbon tails in synthetic PS molecules were investigated. DimyristoylPS (C14: 0), dipalmitoyl-PS (C16: 0), and distearoyl-PS (18: 0) did not reduce IFN-c production. Interestingly, distearoyl-PS (18: 0) increased the IFN-c production in
Fig. 4. Association and localization of phospholipids. Splenic naı¨ve T cells were incubated with NBD-labeled phospholipids at 37 C for 1 h. The cells were washed with ice-cold PBS and stained with Quantum Red-labeled anti-Thy1.2 mAb at 4 C for 30 min. After washing, the cells were fixed with 4% paraformaldehyde–PBS and the association and localization of NBD-labeled phospholipids examined using flow cytometry (A) or confocal microscopy (B).
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Fig. 5. Effects of molecular structures of PS. Splenic naı¨ve T cells were incubated for 18 h with Con A (2.5 lg/ml) in the absence or presence of (A) dimyristoyl-PS (C14: 0) and dipalmitoyl-PS (C16: 0), or distearoyl-PS (18: 0), (B) distearoyl-PS (18: 0), dioleoyl-PS (C18: 1) or dilinoleoyl-PS (C18: 2), and (C) natural PS or oleoyl-lyso-PS (C18: 1). The supernatants of splenic T cell cultures were collected and IFN-c levels were measured as described under Materials and methods. The values are means ± SD of triplicate cultures from three independent experiments.
a dose-dependent manner (Fig. 5A). Furthermore, the effect of the number of unsaturated bonds of hydrophobic hydrocarbon tails in the PS molecule was investigated. Dioleoyl-PS (18: 1) or dilinoleoyl-PS (C18: 2) reduced the IFN-c production in a dose-dependent manner, but distearoyl-PS (C18: 0) did not. The inhibitory effect on IFN-c production by dilinoleoyl-PS (C18: 2) was higher than that of oleoyl-PS (C18: 1) (Fig. 5B). These results suggest that the number of unsaturated bonds of hydrophobic hydrocarbon tails in the PS molecule is critical for the inhibitory effect of IFN-c production by PS. It is likely that oleoyl-PS (C18: 1) is the main component of IFN-c inhibitory effects by PS, produced by human D1-DMBA-3 tumor cells. The PS molecule has two hydrophobic hydrocarbon tails. Phospholipase A hydrolyzes 1- or 2-fatty acid tails and converts PS into lyso-PS, which has one hydrophobic hydrocarbon tail. Lyso-PS stimulates cell signaling and plays important biological roles in humans and other animals. We investigated the effect of lyso-PS on IFN-c production. Treatment of splenic T cells with lyso-PS (C18: 1) caused the inhibition of IFN-c production. A higher concentration of lyso-PS was required to inhibit IFN-c production compared with PS (Fig. 5C). Recent reports [20] suggest that lyso-PS-treated CHO-K1 cells show different morphological features from those treated with PS: cells incubated with lyso-PS underwent extensive membrane fragmentation and swelling, while those incubated with PS became round and underwent a dramatic reduction of cellular volume. In our system, the morphological features of lyso-PS-treated T cells were different from those treated with PS (data not shown). The molecular mechanism in the IFN-c inhibitory effect by lyso-PS is probably not equal to that of PS, and lyso-PS may not contribute to the IFN-c inhibitory effects by PS. Collectively, IFN-c inhibitory effects by PS are highly dependent on the molecular structure of PS.
Effects of PS on IFN-c mRNA expression The effect of the addition time of PS on the IFN-c production in splenic T cells in response to Con A stimulation was investigated, and the complete inhibition of IFN-c production was observed when PS was added concurrently with Con A (Fig. 6). The addition of PS 1, 3, 6 or 12 h after Con
Fig. 6. Effect of PS addition time on Con A-induced IFN-c production. PS (50 lg/ml) was added to a splenic naı¨ve T cell culture at the indicted time after activation with Con A (2.5 lg/ml). The supernatants of splenic naı¨ve T cell cultures were collected and IFN-c levels were measured as described under Materials and methods. The values are means ± SD of triplicate cultures from three independent experiments.
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A stimulation also showed an inhibitory effect on IFN-c production, and the intensity of the inhibitory effect became lower in the above time order of PS addition. This result suggests that the potency of PS results in the suppression of IFN-c production by decreasing the stability of IFN-c mRNA. Therefore, we investigated whether PS affected the stability of IFN-c mRNA using an RT-PCR assay. Con A-induced IFN-c mRNA expression was increased in
a time-dependent manner (Fig. 7A). IFN-c production was also increased (Fig. 7B). Actinomycin D, a transcriptional inhibitor, inhibited IFN-c mRNA transcription in splenic T cells stimulated with Con A (Fig. 7C). As shown in Fig. 7D, the addition of PS brought about a 40% reduction in IFN-c mRNA expression in Con A-stimulated splenic T cells treated with actinomycin D. These results suggest that PS decreases the stability of IFN-c mRNA.
Fig. 7. Effects of PS on the expression of IFN-c mRNA. (A) Time course of IFN-c mRNA expression. Splenic naı¨ve T cells were incubated with Con A (2.5 lg/ml) and the cells were collected at the indicated time. (B) Kinetics of IFN-c secretion induced by Con A. Splenic naı¨ve T cells were incubated with Con A (2.5 lg/ml) for the indicated times and IFN-c concentrations in the supernatant were measured using ELISA. (C) Effect of actinomycin D on IFN-c mRNA expression. Splenic naı¨ve T cells were incubated with Con A (2.5 lg/ml) and after 12 h, actinomycin D (1 lg/ml) was added. The cells were collected at the indicated time. (D) Effect of PS on the stability of IFN-c mRNA. Splenic naı¨ve T cells were incubated with Con A (2.5 lg/ml) and after 12 h, actinomycin D (1 lg/ml) and PS (50 lg/ml) were added. After 1 h, the cells were collected, total RNAs were extracted, and RT-PCR was performed, as described under Materials and methods.
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In conclusion, the differences in the polar head group and the length and saturation of the two hydrophobic hydrocarbon tails in phospholipid molecules make a difference in the effect to IFN-c production in T cells. PS, which has a negatively charged polar head group and two C18hydrophobic hydrocarbon tails containing unsaturated bonds, completely inhibits IFN-c production in mouse T cells. In addition, the suppression of IFN-c production in T cells by PS is involved in decreasing the stability of IFN-c mRNA. At present, it is not clear how PS decrease the stability of IFN-c mRNA. Acknowledgments We are grateful to Mr. K. Saegusa, Miss M. Mizutani, Miss N. Shouzi, Miss. M. Itou, and Miss Y. Inaba for technical assistance. References [1] Y. Nishizuka, Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C, Science 258 (1992) 607–614. [2] R.M. Bell, D.J. Burns, Lipid activation of protein kinase C, J. Biol. Chem. 266 (1991) 4661–4664. [3] K. Igarashi, M. Kaneda, A. Yamaji, T.C. Saido, U. Kikkawa, Y. Ono, K. Inoue, M. Umeda, A novel phosphatidylserine-binding peptide motif defined by an anti-idiotypic monoclonal antibody. Localization of phosphatidylserine-specific binding sites on protein kinase C and phosphatidylserine decarboxylase, J. Biol. Chem. 270 (1995) 29075–29078. [4] K.G. Mann, R.J. Jenny, S. Krishnaswamy, Cofactor proteins in the assembly and expression of blood clotting enzyme complexes, Annu. Rev. Biochem. 57 (1988) 915–956. [5] P. Comfurius, E.F. Smeets, G.M. Willems, E.M. Bevers, R.F. Zwaal, Assembly of the prothrombinase complex on lipid vesicles depends on the stereochemical configuration of the polar headgroup of phosphatidylserine, Biochemistry 33 (1994) 10319–10324. [6] F.M. Stekhoven, J. Tijmes, M. Umeda, K. Inoue, J.J. De Pont, Monoclonal antibody to phosphatidylserine inhibits Na+/K(+)ATPase activity, Biochim. Biophys. Acta 1194 (1994) 155–165. [7] F. Sakane, K. Yamada, S. Imai, H. Kanoh, Porcine 80-kDa diacylglycerol kinase is a calcium-binding and calcium/phospholipid-dependent enzyme and undergoes calcium-dependent translocation, J. Biol. Chem. 266 (1991) 7096–7100.
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[8] S. Ghosh, W.Q. Xie, A.F. Quest, G.M. Mabrouk, J.C. Strum, R.M. Bell, The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras, J. Biol. Chem. 269 (1994) 10000–10007. [9] P.L. Tuma, M.C. Stachniak, C.A. Collins, Activation of dynamin GTPase by acidic phospholipids and endogenous rat brain vesicles, J. Biol. Chem. 268 (1993) 17240–17246. [10] J. Gagne, C. Giguere, G. Tocco, M. Ohayon, R.F. Thompson, M. Baudry, G. Massicotte, Effect of phosphatidylserine on the binding properties of glutamate receptors in brain sections from adult and neonatal rats, Brain Res. 740 (1996) 337–345. [11] K. Schroder, P.J. Hertzog, T. Ravasi, D.A. Hume, Interferongamma: an overview of signals, mechanisms and functions, J. Leukoc. Biol. 75 (2004) 163–189. [12] M.E. Handel-Fernandez, X. Cheng, L.M. Herbert, D.M. Lopez, Down-regulation of IL-12, not a shift from a T helper-1 to a T helper-2 phenotype, is responsible for impaired IFN-gamma production in mammary tumor-bearing mice, J. Immunol. 158 (1997) 280–286. [13] X. Cheng, D.M. Lopez, CD4+, but not CD8+, T cells from mammary tumor-bearing mice have a down-regulated production of IFNgamma: role of phosphatidyl serine, J. Immunol. 160 (1998) 2735– 2741. [14] T. Kakiuchi, A. Takatsuki, M. Watanabe, H. Nariuchi, Inhibition by brefeldin A of the specific B cell antigen presentation to MHC class II-restricted T cells, J. Immunol. 147 (1991) 3289– 3295. [15] T.R. Mosmann, R.L. Coffman, TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties, Annu. Rev. Immunol. 7 (1989) 145–173. [16] X. Ma, A. DÕAndrea, M. Kubin, M. Aste-Amezaga, A. Sartori, J. Monteiro, L. Showe, M. Wysocka, G. Trinchieri, Production of interleukin-12, Res. Immunol. 146 (1995) 432–438. [17] A. DÕAndrea, M. Rengaraju, N.M. Valiante, J. Chehimi, M. Kubin, M. Aste, S.H. Chan, M. Kobayashi, D. Young, E. Nickbarg, et al., Production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells, J. Exp. Med. 176 (1992) 1387– 1398. [18] T. Kobayashi, Y. Arakawa, Transport of exogenous fluorescent phosphatidylserine analogue to the Golgi apparatus in cultured fibroblasts, J. Cell Biol. 113 (1991) 235–244. [19] C. Calderon, Z.H. Huang, D.A. Gage, E.M. Sotomayor, D.M. Lopez, Isolation of a nitric oxide inhibitor from mammary tumor cells and its characterization as phosphatidyl serine, J. Exp. Med. 180 (1994) 945–958. [20] K. Uchida, K. Emoto, D.L. Daleke, K. Inoue, M. Umeda, Induction of apoptosis by phosphatidylserine, J. Biochem. (Tokyo) 123 (1998) 1073–1078.
Negatively charged phospholipids suppress IFN-c production in T cells Satoshi Yotsumoto a, Terutaka Kakiuchi b, Yukihiko Aramaki a,* a
School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan b Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, Tokyo 143-8540, Japan Received 25 October 2005 Available online 7 November 2005
Abstract The effect of phospholipids on IFN-c production in mouse T cells was investigated. Phosphatidylserine (PS), which has a negatively charged head group, completely inhibited IFN-c production in splenic naı¨ve T cells and antigen-dependent IFN-c production in Th1 clone 42-6A cells, whereas other phospholipids, which have neutrally charged head group, had no effect. The structural requirements for IFN-c inhibitory effects by PS were investigated, and dimyristoyl-PS (C14: 0) and dipalmitoyl-PS (C16: 0) had no effect on IFN-c production, and interestingly, distearoyl-PS (18: 0) increased IFN-c production. Dioleoyl-PS (C18: 1), dilinoleoyl-PS (C18: 2), and oleoyl-lyso-PS (C18: 1) completely inhibited IFN-c production. To clarify this mechanism, we focused on the stability of IFN-c mRNA, and the treatment of splenic naı¨ve T cells with PS brought about 40% reductions in IFN-c mRNA expression in the presence of actinomycin D. Collectively, IFN-c inhibitory effects by PS are highly dependent on the molecular structure of PS and involve the decreasing of the stability of IFN-c mRNA. 2005 Elsevier Inc. All rights reserved. Keywords: Phospholipids; T cell; IFN-c; Phosphatidylserine; Molecular structure
Phospholipids in the mammalian plasma membrane consist of various phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, phosphatidylserine (PS), phosphatidic acid, and phosphatidylinositol. Phospholipid molecules have a polar head group and two hydrophobic hydrocarbon tails. The tails are fatty acids and can differ in length. The tails usually have one or more cis-double bonds (unsaturated bonds). PS is a major anionic phospholipid component of mammalian cell membranes. Recent studies have shown that PS is an essential cofactor for the activation of protein kinase C [1–3] and blood coagulation [4,5]. PS has also been shown to regulate the activities of various enzymes, such as Na+/K+-ATPase [6], diacylglycerol kinase [7], B-Raf protein kinase [8], dynamin GTPase [9], and glutamate [10]. IFN-c, mainly produced by T cells and NK cells, is a multifunctional cytokine known to be a regulator of numerous immunological functions. Especially, IFN-c plays an impor*
Corresponding author. Fax: +81 426 76 3183. E-mail address: [email protected] (Y. Aramaki).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.10.179
tant role in the elimination of tumor cells by cytotoxic T lymphocytes. It is well known that IFN-c production is dramatically reduced in T cells from mice bearing large mammary tumors [11]. A recent report suggested that various phospholipids were produced by a D1-DMBA-3 mammary tumor and may contribute to the down-regulation of IFN-c production in T cells from mice bearing large D1DMBA-3 mammary tumors [12,13]. However, it is not clear that how the molecular structure of phospholipids is important for the IFN-c inhibitory effect. In this paper, we investigated the effect of the molecular structure of phospholipids on IFN-c production in mouse T cells. The findings suggested that negatively charged phospholipids inhibited IFN-c production and that the inhibitory effect is highly dependent on the molecular structure of negatively charged phospholipids. Materials and methods Materials. Phosphatidylcholine (PC) was purchased from Nippon Oil and Fat (Tokyo, Japan). Ovalbumin (OVA), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidyl-
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serine (PS), and Quantum Red-labeled anti-Thy1.2 monoclonal antibody were purchased from Sigma Chemical (St. Louis, MO). Anti-mouse CD3 e monoclonal antibody and recombinant mouse IL-2 were obtained from Genzyme (Cambridge, MA). Dimyristoyl-PS (C14: 0), dipalmitoyl-PS (C16: 0), distearoyl-PS (18: 0), dioleoyl-PS (C18: 1), dilinoleoyl-PS (C18: 2), and oleoyl-lyso-PS (C18: 1 were purchased from Avanti Polar Lipids (Alabaster, AL). Animals. BALB/c mice were purchased from Japan SLC (Shizuoka, Japan). All mice were used at 6–10 weeks of age. Animal use and relevant experimental procedures were approved by the Tokyo University of Pharmacy and Life Science Committee on the care and use of laboratory animals. Preparation of splenic naı¨ve T cells. Splenic naı¨ve T cells were purified by passing spleen cells through nylon wool columns twice. After washing twice with PBS, the cells were suspended in RPMI1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.28% NaHCO3, 10 mM Hepes, 5 · 105 M of 2-mercaptoethanol, 100 U/ml penicillin, and 100 lg/ml streptomycin in a humidified 5% CO2 at 37 C. Cell purity was ascertained by flow cytometric analysis using FACScalibur (Becton– Dickinson, Mountain View, CA). Routinely, the naı¨ve T cell preparations had a purity of 90–95%. T cell clones. Ovalbumin (OVA)-specific Th1 clones, 42-6A cells established from BALB/c mice [14] were maintained in RPMI1640 medium supplemented with 10 U/ml murine recombinant IL-2. Preparation of splenic adherent cells. Spleen cells from BALB/c mice were incubated in 100-mm plastic dishes (Iwaki glass, Chiba, Japan) for 90 min at 37 C in RPMI1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.28% NaHCO3, 10 mM Hepes, 50 lM of 2mercaptoethanol, 100 U/ml penicillin, and 100 lg/ml streptomycin. Nonadherent cells were removed by washing the dishes five times with icecold PBS. Splenic adherent cells (SAC) were recovered by 0.5% lidocaine– PBS (Astrazeneca, Osaka, Japan). SAC were washed twice and resuspended in RPMI1640 medium. SAC used as antigen presenting cells for OVA-specific Th1 clones, 42-6A cells. Preparation of phospholipid suspension. Phospholipids in chloroform solution were dried in a test tube under N2 gas and desiccated under vacuum for at least 2 h. The dry lipid films were hydrated by the addition of 1 ml of 10 mM Tris-buffer saline (pH 7.2) and the phospholipid suspension was prepared. The suspensions were then sterilized by passing through a membrane filter (0.45 lm; Iwaki, Tokyo, Japan). Determination of murine IFN-c. The levels of IFN-c in culture supernatants were routinely assayed by sandwich ELISA using pairs of purified
A
capture and biotinylated detection monoclonal antibodies recognizing murine IFN-c, according to the manufacturerÕs protocols. Association of phospholipids. Splenic T cells were incubated with NBDlabeled phospholipids at 37 C for 1 h. The cells were washed with ice-cold PBS and stained with Quantum Red-labeled anti-Thy1.2 mAb at 4 C for 30 min. After washing, the cells were fixed with 4% paraformaldehyde– PBS, and the association and localization of NBD-phospholipids were examined using flow cytometry or confocal microscopy, respectively. RT-PCR analysis. IFN-c mRNA expressions were determined using RT-PCR. Total RNA was isolated from splenic naı¨ve T cells with Isogen solution (Nippon Gene, Toyama, Japan). cDNAs were synthesized using SuperScript. cDNA were then amplified with specific primers for IFN-c. Each primer was designed based on the mouse sequences (IFN-c—forward, 5 0 -GACTTCAAAGATCTGAGG-3; IFN-c—reverse, 5 0 -AACGCT ACACACTGCATCTTGG-3 0 ; b-actin—forward, 5 0 -GCACCACACCTT CTACAATGA-G-3 0 ; b-actin—reverse, 5 0 -TTGGCATAGAGGTCTTTA CGGA-3 0 ). PCR was performed for 26 cycles of denaturation at 94 C for 60 s, annealing at 55 C for 60 s, and extension at 72 C for 60 s. The amplified products were analyzed on 2% agarose gels containing 0.1 lg/ml ethidium bromide. The band intensity was analyzed with NIH Image.
Results and discussion IFN-c production in splenic T cells is inhibited by negatively charged phospholipids We investigated the effects of various natural phospholipids on IFN-c production in mouse splenic T cells. As shown in Fig. 1A, IFN-c production elicited by the mitogen, 5 lg/ml concanavalin A (Con A), was inhibited when splenic T cells were treated with PS and PA (each 50 lg/ ml), which have a negatively polar head group. No inhibitory effects were observed in PC, PI, and PE (50 lg/ml), which have a neutral head group. PS and PA showed dose-dependent inhibitory effects on IFN-c production, and complete inhibition was observed in PS at 50 lg/ml and in PA at 200 lg/ml (Fig. 1B). A higher concentration of PA was required to inhibit IFN-c production compared
B
Fig. 1. Effects of various phospholipids on IFN-c production. (A) Splenic naı¨ve T cells were incubated for 18 h with Con A (2.5 lg/ml) in the presence of PC, PI, PE, PS or PA (50 lg/ml). (B) Effects of PS or PA doses on IFN-c production. Splenic naı¨ve T cells were incubated for 18 h with Con A (2.5 lg/ml) in the presence of PS (0–100 lg/ml) or PA (0–200 lg/ml). The supernatants of splenic T cell cultures were collected and IFN-c levels were measured as described under Materials and methods. The values are means ± SD of triplicate cultures from three independent experiments.
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with that of PS. The viability of splenic T cells after PS treatment was estimated with a trypan blue dye exclusion test, and no changes in viability were observed at the concentrations used (data not shown). These results suggest that phospholipids, which have a negatively charged polar head group, inhibit IFN-c production in splenic T cells without a reduction in cell viability. Effect of PS on IFN-c production induced by various stimulations The effects of PS on IFN-c production in splenic T cells elicited by various stimulants were investigated. As shown in Fig. 2, IFN-c production in splenic T cells elicited by anti-CD3e mAb (10 lg/ml), or 50 ng/ml phorbol ester plus ionomycin (500 ng/ml), was also completely inhibited by the treatment with PS (50 lg/ml). PC did not affect the IFN-c production. These results indicate that PS has the ability to inhibit IFN-c production in splenic T cells irrespective of the presence of stimulants. Effect of PS on antigen-specific IFN-c production in Th1 clone cells Naı¨ve CD4+ T cells differentiate into T helper 1 (Th1) or Th2 cells depending upon the cytokine environment. Th1 cells produce IL-2 and IFN-c, whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13 [15]. It is well known that IFN-c production by Th1 cells is induced when the cells are cocultured with antigen presenting cells (APC), such as macrophage/dendritic cells, in the presence of antigens (designated as antigen-dependent IFN-c production) [16,17]. Next, we investigated the effect of PS on antigendependent IFN-c production using OVA-specific Th1 clone 42-6A cells. As shown in Fig. 3, antigen-dependent IFN-c production in 42-6A cells, which were cocultured with SAC in the presence of 2 mg/ml OVA, was completely
Fig. 3. Effect of PS on antigen-dependent IFN-c production. The coculture of 42-6A cells with SAC was incubated for 18 h with OVA (2 mg/ml) in the presence of PC or PS. The supernatants of 42-6A cell cultures were collected and IFN-c levels were measured, as described under Materials and methods. The values are means ± SD of triplicate cultures from three independent experiments.
inhibited by PS in a dose-dependent manner, but not PC. Xiaofang et al. previously reported that PS effectively down-regulated the IFN-c production in naı¨ve CD4+ T cells from the spleen in vitro [13]. In this study, we indicate that PS inhibits antigen-dependent IFN-c production from differentiated T cells, such as CD4+ Th1 cells. Studies are now underway to clarify whether IFN-c production in CD8+ cytotoxic T lymphocytes was inhibited by PS. Association and localization of PS To clarify whether the inhibition of IFN-c production by phospholipids resulted from differences in the amount of phospholipids taken up, the association (sum of bind-
Fig. 2. Effects of PS on IFN-c production in splenic naı¨ve T cells with various stimulations. Splenic naı¨ve T cells were incubated for 18 h with anti-CD3e mAb (10 lg/ml), or PMA (50 ng/ml) plus ionomycin (500 ng/ml) in the presence of PC or PS (0–50 lg/ml). The supernatants of splenic T cell cultures were collected and the IFN-c levels were measured as described under Materials and methods. The values are means ± SD of triplicate cultures from three independent experiments.
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ing and uptake) of phospholipids was studied using NBDlabeled phospholipids. NBD is a fluorescence probe and the association of phospholipids can easily be evaluated using flow cytometry and confocal laser scanning microscopy. As shown in Fig. 4A, the fluorescence of cells incubated with NBD-labeled PS (NBD-PS) was observed, but that of NBD-PC was not. Furthermore, NBD-PS was not colocalized with Quantum Red-labeled monoclonal antibody against Thy1.2, a cell surface marker of T cells (Fig. 4B). These results suggest that the difference in the amount of phospholipids associated with T cells was involved in the induction of IFN-c inhibitory effects and PS was not localized to the cell surface. A previous report [18] suggested that exogenous PS is mainly transported to the Golgi apparatus by three consecutive processes in CHO-K1 cells. They are: (a) the insertion of PS into the outer leaflet of the plasma membrane, (b) translocation of PS from the outer to the inner leaflet of the plasma membrane, and (c) transport of PS from the inner leaflet
of the plasma membrane to the Golgi apparatus via a nonvesicular pathway. It is likely that PS, localized in inner leaflet of the plasma membrane or Golgi apparatus, affects T cell function. Structure–activity relationship of IFN-c inhibitory effects by PS Stearoyl-PS (18: 0) and oleoyl-PS (C18: 1) are major components of PS produced by human D1-DMBA-3 tumor cells [19]. However, it is not clear that how the molecular structure of PS is important for IFN-c inhibitory effects by PS. We thus next examined the structural requirements for IFN-c inhibitory effects by PS. First, the effects of the length of hydrophobic hydrocarbon tails in synthetic PS molecules were investigated. DimyristoylPS (C14: 0), dipalmitoyl-PS (C16: 0), and distearoyl-PS (18: 0) did not reduce IFN-c production. Interestingly, distearoyl-PS (18: 0) increased the IFN-c production in
Fig. 4. Association and localization of phospholipids. Splenic naı¨ve T cells were incubated with NBD-labeled phospholipids at 37 C for 1 h. The cells were washed with ice-cold PBS and stained with Quantum Red-labeled anti-Thy1.2 mAb at 4 C for 30 min. After washing, the cells were fixed with 4% paraformaldehyde–PBS and the association and localization of NBD-labeled phospholipids examined using flow cytometry (A) or confocal microscopy (B).
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Fig. 5. Effects of molecular structures of PS. Splenic naı¨ve T cells were incubated for 18 h with Con A (2.5 lg/ml) in the absence or presence of (A) dimyristoyl-PS (C14: 0) and dipalmitoyl-PS (C16: 0), or distearoyl-PS (18: 0), (B) distearoyl-PS (18: 0), dioleoyl-PS (C18: 1) or dilinoleoyl-PS (C18: 2), and (C) natural PS or oleoyl-lyso-PS (C18: 1). The supernatants of splenic T cell cultures were collected and IFN-c levels were measured as described under Materials and methods. The values are means ± SD of triplicate cultures from three independent experiments.
a dose-dependent manner (Fig. 5A). Furthermore, the effect of the number of unsaturated bonds of hydrophobic hydrocarbon tails in the PS molecule was investigated. Dioleoyl-PS (18: 1) or dilinoleoyl-PS (C18: 2) reduced the IFN-c production in a dose-dependent manner, but distearoyl-PS (C18: 0) did not. The inhibitory effect on IFN-c production by dilinoleoyl-PS (C18: 2) was higher than that of oleoyl-PS (C18: 1) (Fig. 5B). These results suggest that the number of unsaturated bonds of hydrophobic hydrocarbon tails in the PS molecule is critical for the inhibitory effect of IFN-c production by PS. It is likely that oleoyl-PS (C18: 1) is the main component of IFN-c inhibitory effects by PS, produced by human D1-DMBA-3 tumor cells. The PS molecule has two hydrophobic hydrocarbon tails. Phospholipase A hydrolyzes 1- or 2-fatty acid tails and converts PS into lyso-PS, which has one hydrophobic hydrocarbon tail. Lyso-PS stimulates cell signaling and plays important biological roles in humans and other animals. We investigated the effect of lyso-PS on IFN-c production. Treatment of splenic T cells with lyso-PS (C18: 1) caused the inhibition of IFN-c production. A higher concentration of lyso-PS was required to inhibit IFN-c production compared with PS (Fig. 5C). Recent reports [20] suggest that lyso-PS-treated CHO-K1 cells show different morphological features from those treated with PS: cells incubated with lyso-PS underwent extensive membrane fragmentation and swelling, while those incubated with PS became round and underwent a dramatic reduction of cellular volume. In our system, the morphological features of lyso-PS-treated T cells were different from those treated with PS (data not shown). The molecular mechanism in the IFN-c inhibitory effect by lyso-PS is probably not equal to that of PS, and lyso-PS may not contribute to the IFN-c inhibitory effects by PS. Collectively, IFN-c inhibitory effects by PS are highly dependent on the molecular structure of PS.
Effects of PS on IFN-c mRNA expression The effect of the addition time of PS on the IFN-c production in splenic T cells in response to Con A stimulation was investigated, and the complete inhibition of IFN-c production was observed when PS was added concurrently with Con A (Fig. 6). The addition of PS 1, 3, 6 or 12 h after Con
Fig. 6. Effect of PS addition time on Con A-induced IFN-c production. PS (50 lg/ml) was added to a splenic naı¨ve T cell culture at the indicted time after activation with Con A (2.5 lg/ml). The supernatants of splenic naı¨ve T cell cultures were collected and IFN-c levels were measured as described under Materials and methods. The values are means ± SD of triplicate cultures from three independent experiments.
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A stimulation also showed an inhibitory effect on IFN-c production, and the intensity of the inhibitory effect became lower in the above time order of PS addition. This result suggests that the potency of PS results in the suppression of IFN-c production by decreasing the stability of IFN-c mRNA. Therefore, we investigated whether PS affected the stability of IFN-c mRNA using an RT-PCR assay. Con A-induced IFN-c mRNA expression was increased in
a time-dependent manner (Fig. 7A). IFN-c production was also increased (Fig. 7B). Actinomycin D, a transcriptional inhibitor, inhibited IFN-c mRNA transcription in splenic T cells stimulated with Con A (Fig. 7C). As shown in Fig. 7D, the addition of PS brought about a 40% reduction in IFN-c mRNA expression in Con A-stimulated splenic T cells treated with actinomycin D. These results suggest that PS decreases the stability of IFN-c mRNA.
Fig. 7. Effects of PS on the expression of IFN-c mRNA. (A) Time course of IFN-c mRNA expression. Splenic naı¨ve T cells were incubated with Con A (2.5 lg/ml) and the cells were collected at the indicated time. (B) Kinetics of IFN-c secretion induced by Con A. Splenic naı¨ve T cells were incubated with Con A (2.5 lg/ml) for the indicated times and IFN-c concentrations in the supernatant were measured using ELISA. (C) Effect of actinomycin D on IFN-c mRNA expression. Splenic naı¨ve T cells were incubated with Con A (2.5 lg/ml) and after 12 h, actinomycin D (1 lg/ml) was added. The cells were collected at the indicated time. (D) Effect of PS on the stability of IFN-c mRNA. Splenic naı¨ve T cells were incubated with Con A (2.5 lg/ml) and after 12 h, actinomycin D (1 lg/ml) and PS (50 lg/ml) were added. After 1 h, the cells were collected, total RNAs were extracted, and RT-PCR was performed, as described under Materials and methods.
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In conclusion, the differences in the polar head group and the length and saturation of the two hydrophobic hydrocarbon tails in phospholipid molecules make a difference in the effect to IFN-c production in T cells. PS, which has a negatively charged polar head group and two C18hydrophobic hydrocarbon tails containing unsaturated bonds, completely inhibits IFN-c production in mouse T cells. In addition, the suppression of IFN-c production in T cells by PS is involved in decreasing the stability of IFN-c mRNA. At present, it is not clear how PS decrease the stability of IFN-c mRNA. Acknowledgments We are grateful to Mr. K. Saegusa, Miss M. Mizutani, Miss N. Shouzi, Miss. M. Itou, and Miss Y. Inaba for technical assistance. References [1] Y. Nishizuka, Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C, Science 258 (1992) 607–614. [2] R.M. Bell, D.J. Burns, Lipid activation of protein kinase C, J. Biol. Chem. 266 (1991) 4661–4664. [3] K. Igarashi, M. Kaneda, A. Yamaji, T.C. Saido, U. Kikkawa, Y. Ono, K. Inoue, M. Umeda, A novel phosphatidylserine-binding peptide motif defined by an anti-idiotypic monoclonal antibody. Localization of phosphatidylserine-specific binding sites on protein kinase C and phosphatidylserine decarboxylase, J. Biol. Chem. 270 (1995) 29075–29078. [4] K.G. Mann, R.J. Jenny, S. Krishnaswamy, Cofactor proteins in the assembly and expression of blood clotting enzyme complexes, Annu. Rev. Biochem. 57 (1988) 915–956. [5] P. Comfurius, E.F. Smeets, G.M. Willems, E.M. Bevers, R.F. Zwaal, Assembly of the prothrombinase complex on lipid vesicles depends on the stereochemical configuration of the polar headgroup of phosphatidylserine, Biochemistry 33 (1994) 10319–10324. [6] F.M. Stekhoven, J. Tijmes, M. Umeda, K. Inoue, J.J. De Pont, Monoclonal antibody to phosphatidylserine inhibits Na+/K(+)ATPase activity, Biochim. Biophys. Acta 1194 (1994) 155–165. [7] F. Sakane, K. Yamada, S. Imai, H. Kanoh, Porcine 80-kDa diacylglycerol kinase is a calcium-binding and calcium/phospholipid-dependent enzyme and undergoes calcium-dependent translocation, J. Biol. Chem. 266 (1991) 7096–7100.
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