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Roles of Glycosylphosphatidylinositols of Toxoplasma gondii

2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês

10.1074/jbc.m304791200

1083-351X

Françoise Debierre-Grockiego, Nahid Azzouz, Jörg Schmidt, Jean‐François Dubremetz, Hildegard Geyer, Rudolf Geyer, Ralf Weingart, Richard R. Schmidt, Ralph Τ. Schwarz,

Parasitic Infections and Diagnostics

Toxoplasma gondii is a ubiquitous parasitic protozoan, which causes congenital infectious diseases as well as severe encephalitis, a major cause of death among immune-deficient persons, such as AIDS patients. T. gondii is normally controlled by the immune system of healthy individuals, leading to an asymptomatic infection. T. gondii triggers early cytokine production, which, to a certain extent, protects the host against replication of tachyzoites, the infective form of the parasite. Glycosylphosphatidylinositols (GPIs) constitute a class of glycolipids that have various functions, the most fundamental being to link proteins to eucaryotic cell membranes. GPIs are involved in the pathogenicity of other protozoan parasites and are known to induce tumor necrosis factor-α (TNFα) production. We show that GPIs highly purified from T. gondii tachyzoites, as well as their core glycans, induce TNFα production in macrophages. A chemically synthesized GPI of T. gondii lacking its lipid moiety, GPIa, has the same effect as the natural GPIs, whereas a chemically synthesized molecule with dialkylglycerol instead of diacylglycerol as lipid moiety, GPIb, does not induce TNFα production. Moreover, GPIb inhibits the TNFα production induced by T. gondii GPI or by GPIa. The core glycan prepared from the two chemically synthesized molecules activates macrophages, showing that the lipid moiety may regulate signaling. Stimulation of macrophages with GPIs of T. gondii results in activation of the transcription factor NF-κB, which is inhibited by the chemically synthesized GPIb, suggesting the involvement of NF-κB in TNFα gene expression. Our results support the idea that T. gondii GPIs are bioactive factors that participate in the production of TNFα during toxoplasmal pathogenesis. Toxoplasma gondii is a ubiquitous parasitic protozoan, which causes congenital infectious diseases as well as severe encephalitis, a major cause of death among immune-deficient persons, such as AIDS patients. T. gondii is normally controlled by the immune system of healthy individuals, leading to an asymptomatic infection. T. gondii triggers early cytokine production, which, to a certain extent, protects the host against replication of tachyzoites, the infective form of the parasite. Glycosylphosphatidylinositols (GPIs) constitute a class of glycolipids that have various functions, the most fundamental being to link proteins to eucaryotic cell membranes. GPIs are involved in the pathogenicity of other protozoan parasites and are known to induce tumor necrosis factor-α (TNFα) production. We show that GPIs highly purified from T. gondii tachyzoites, as well as their core glycans, induce TNFα production in macrophages. A chemically synthesized GPI of T. gondii lacking its lipid moiety, GPIa, has the same effect as the natural GPIs, whereas a chemically synthesized molecule with dialkylglycerol instead of diacylglycerol as lipid moiety, GPIb, does not induce TNFα production. Moreover, GPIb inhibits the TNFα production induced by T. gondii GPI or by GPIa. The core glycan prepared from the two chemically synthesized molecules activates macrophages, showing that the lipid moiety may regulate signaling. Stimulation of macrophages with GPIs of T. gondii results in activation of the transcription factor NF-κB, which is inhibited by the chemically synthesized GPIb, suggesting the involvement of NF-κB in TNFα gene expression. Our results support the idea that T. gondii GPIs are bioactive factors that participate in the production of TNFα during toxoplasmal pathogenesis. Toxoplasma gondii is an obligate intracellular protozoan parasite that invades nearly all nucleated cells in a complex multistep process (1Joiner K.A. Dubremetz J.F. Infect. Immun. 1993; 61: 1169-1172Crossref PubMed Google Scholar). Primo-infection of pregnant women may cause congenital toxoplasmosis with the risk of severe injury of the fetus (2Remington J.S. Desmonts G. Remington J.S. Klein J.O. Infectious Diseases of the Fetus and Newborn Infant. W. B. Saunders Co., Philadelphia1990: 89-195Google Scholar). The reactivation of previously dormant brain cysts containing the bradyzoite stage leads to Toxoplasma encephalitis, a major cause of death among immune-deficient persons (3Luft B.J. Remington J.S. Clin. Infect. Dis. 1992; 15: 211-222Crossref PubMed Scopus (1044) Google Scholar). Toxoplasmosis in general is a benign infection that causes mild or even no symptoms in immunocompetent individuals. A strong cell-mediated immunity induced by T. gondii protects the host against rapid parasite growth and consequent pathologies. This immunity is associated with the development of CD4+ and CD8+ T lymphocytes producing type 1 cytokines, in particular interferon γ (IFNγ) 1The abbreviations used are: IFNγ, interferon γ; GPIs, glycosylphosphatidylinositols; TNFα, tumor necrosis factor-α; IL, interleukin; LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay; TLRs, Toll-like receptors.1The abbreviations used are: IFNγ, interferon γ; GPIs, glycosylphosphatidylinositols; TNFα, tumor necrosis factor-α; IL, interleukin; LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay; TLRs, Toll-like receptors. (4Gazzinelli R.T. Denkers E.Y. Sher A. Infect. Agents Dis. 1993; 2: 139-149PubMed Google Scholar). Expression of interleukin (IL)-1β, IL-2, IL-12, and tumor necrosis factor-α (TNFα) mRNAs was shown in the brain and the spleen of infected mice (5Hunter C.A. Roberts C.W. Alexander J. Eur. J. Immunol. 1992; 22: 2317-2322Crossref PubMed Scopus (93) Google Scholar, 6Deckert-Schluter M. Albrecht S. Hof H. Wiestler O.D. Schluter D. Immunology. 1995; 85: 408-418PubMed Google Scholar, 7Arsenijevic D. Girardier L. Seydoux J. Chang H.R. Dulloo A.G. Am. J. Physiol. 1997; 272: E908-E917PubMed Google Scholar). These cytokines are involved in T cell-independent mechanisms against T. gondii infection. TNFα increases in vitro the number of cysts formed in human fibroblasts, involving the activity of an acidic sphingomyelinase (8Ricard J. Pelloux H. Pathak S. Pipy B. Ambroise-Thomas P. Cell. Signal. 1996; 8: 439-442Crossref PubMed Scopus (19) Google Scholar). TNFα acts synergistically with IFNγ against cerebral toxoplasmosis by inducing indoleamine 2,3-dioxygenase activity, which degrades tryptophan, resulting in an antiparasitic mechanism in brain cells (9Daubener W. Remscheid C. Nockemann S. Pilz K. Seghrouchni S. Mackenzie C. Hadding U. Eur. J. Immunol. 1996; 26: 487-492Crossref PubMed Scopus (78) Google Scholar, 10Daubener W. Spors B. Hucke C. Adam R. Stins M. Kim K.S. Schroten H. Infect. Immun. 2001; 69: 6527-6531Crossref PubMed Scopus (103) Google Scholar). Acute toxoplasmosis that causes rapid death in mice is accompanied by high levels of type 1 cytokines in the serum, including IFNγ and TNFα (11Mordue D.G. Monroy F. La Regina M. Dinarello C.A. Sibley L.D. J. Immunol. 2001; 167: 4574-4584Crossref PubMed Scopus (270) Google Scholar). Although endogenous TNFα is important for resistance against T. gondii, daily treatment of infected mice with TNFα leads to earlier death than in control mice (12Hunter C.A. Suzuki Y. Subauste C.S. Remington J.S. Gross U. Toxoplasma gondii. Vol. 219. Springer-Verlag, Berlin1996: 113-125Crossref Scopus (63) Google Scholar). TNFα mRNA is not expressed in human astrocytoma cells after in vitro infection with different strains of T. gondii (13Pelloux H. Ricard J. Bracchi V. Markowicz Y. Verna J.M. Ambroise-Thomas P. Parasitol. Res. 1994; 80: 271-276Crossref PubMed Scopus (13) Google Scholar), whereas murine inflammatory macrophages are able to synthesize the transcript (14Grunvald E. Chiaramonte M. Hieny S. Wysocka M. Trinchieri G. Vogel S.N. Gazzinelli R.T. Sher A. Infect. Immun. 1996; 64: 2010-2018Crossref PubMed Google Scholar). Infected human monocytes and macrophages do not secrete TNFα (15Pelloux H. Chumpitazi B.F. Santoro F. Polack B. Vuillez J.P. Ambroise-Thomas P. Infect. Immun. 1992; 60: 2672-2676Crossref PubMed Google Scholar), but the cytokine is produced by murine microglial cells and murine macrophages infected with T. gondii (16Fischer H.G. Nitzgen B. Reichmann G. Hadding U. Eur. J. Immunol. 1997; 27: 1539-1548Crossref PubMed Scopus (94) Google Scholar, 17Fischer H.G. Dorfler R. Schade B. Hadding U. Int. Immunol. 1999; 11: 341-349Crossref PubMed Scopus (19) Google Scholar). Treatment of T. gondii-infected monocytes with IFNγ in combination with LPS reduces the invasion index of T. gondii due to the production of TNFα. However, a significant decrease in TNFα secretion is seen in response to IFNγ/LPS in T. gondii-infected monocytes, in comparison with uninfected cells (18Belloni A. Aubert D. Gomez Marin J.E. Le Naour R. Bonhomme A. Guenounou M. Pinon J.M. Parasitol. Res. 2000; 86: 406-412Crossref PubMed Scopus (15) Google Scholar). In addition, active invasion of the parasite is required to mediate the suppression of TNFα production in macrophages (19Butcher B.A. Denkers E.Y. Infect. Immun. 2002; 70: 5216-5224Crossref PubMed Scopus (77) Google Scholar). In the case of stimulation with soluble antigen extract of T. gondii, both murine macrophages (14Grunvald E. Chiaramonte M. Hieny S. Wysocka M. Trinchieri G. Vogel S.N. Gazzinelli R.T. Sher A. Infect. Immun. 1996; 64: 2010-2018Crossref PubMed Google Scholar, 20Gazzinelli R.T. Wysocka M. Hieny S. Scharton-Kersten T. Cheever A. Kuhn R. Muller W. Trinchieri G. Sher A. J. Immunol. 1996; 157: 798-805PubMed Google Scholar, 21Li Z.Y. Manthey C.L. Perera P.Y. Sher A. Vogel S.N. Infect. Immun. 1994; 62: 3434-3440Crossref PubMed Google Scholar) and human neutrophils (22Bliss S.K. Marshall A.J. Zhang Y. Denkers E.Y. J. Immunol. 1999; 162: 7369-7375PubMed Google Scholar) secrete TNFα. Little is known about the mechanisms by which T. gondii induce the production of cytokines. An important reason for determining the molecules responsible for cytokine production relates to their potential use as immunomodulators. The parasite molecules in soluble tachyzoite antigen responsible for TNFα induction are heat stable, partially resistant to proteinase K treatment, and sensitive to periodate oxidation at high concentrations, suggesting that they are protein-associated glycoconjugates (14Grunvald E. Chiaramonte M. Hieny S. Wysocka M. Trinchieri G. Vogel S.N. Gazzinelli R.T. Sher A. Infect. Immun. 1996; 64: 2010-2018Crossref PubMed Google Scholar). Glycosylphosphatidylinositols (GPIs) constitute a class of glycolipids that have various functions, the most fundamental being to link proteins and glycoproteins via their C terminus to cell membrane. Different studies have documented the immunostimulatory and regulatory activities of protozoan GPIs. GPIs of Plasmodium falciparum, Trypanosoma brucei, Leishmania mexicana, and Trypanosoma cruzi are inducers of TNFα production in macrophages (23Schofield L. Hackett F. J. Exp. Med. 1993; 177: 145-153Crossref PubMed Scopus (397) Google Scholar, 24Tachado S.D. Schofield L. Biochem. Biophys. Res. Commun. 1994; 205: 984-991Crossref PubMed Scopus (97) Google Scholar, 25Tachado S.D. Gerold P. Schwarz R. Novakovic S. McConville M. Schofield L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4022-4027Crossref PubMed Scopus (184) Google Scholar, 26Almeida I.C. Camargo M.M. Procopio D.O. Silva L.S. Mehlert A. Travassos L.R. Gazzinelli R.T. Ferguson M.A. EMBO J. 2000; 19: 1476-1485Crossref PubMed Scopus (200) Google Scholar). Free GPIs as well as GPI-anchored proteins are present in T. gondii (27Tomavo S. Schwarz R.T. Dubremetz J.F. Mol. Cell. Biol. 1989; 9: 4576-4580Crossref PubMed Scopus (91) Google Scholar). We show that isolated T. gondii GPIs induce the production of TNFα in macrophages, involving the activation of NF-κB p65, a member of the NF-κB/c-rel family of transcription factors. Materials—[3H]Glucosamine hydrochloride (25 Ci/mmol) was purchased from Amersham Biosciences. Two GPI molecules of T. gondii were chemically synthesized according to Pekari et al. (28Pekari K. Tailler D. Weingart R. Schmidt R.R. J. Org. Chem. 2001; 66: 7432-7442Crossref PubMed Scopus (58) Google Scholar) and have the following structure: GPIa, (ethanolamine-PO4)-Manα1–2Manα1–6(Glc-α1–4GalNAcβ1–4)Manα1–4GlcNα-inositol-PO4, and GPIb, (ethanolamine-PO4)-Manα1–2Manα1–6(Glcα1–4GalNAcβ1–4)Manα1–4GlcNα-inositol-PO4-dialkylglycerol. Dipalmitoylglycerol and distearylglycerol were purchased from Sigma. Parasites and Host Cells—RH strain T. gondii tachyzoites were grown in Vero cells. Confluent cell cultures (75 cm2) were infected with 5 × 107 tachyzoites in Dulbecco's modified Eagle's medium, supplemented with 1% (v/v) fetal calf serum and 2 mm l-glutamine. Cell line and parasites were routinely tested for Mycoplasma contamination. Metabolic Labeling of Tachyzoites—After infection with T. gondii tachyzoites (72 h post-infection), cell cultures were washed twice with glucose-free Dulbecco's modified Eagle's medium containing 20 mm sodium pyruvate. Labeling was performed using the same medium supplemented with 0.5 mCi [3H]glucosamine for 4 h at 37 °C in 5% CO2 atmosphere. After labeling, parasites were released from host cells with 30 strokes in a Dounce homogenizer. Tachyzoites were purified by glass wool filtration as described previously (29Grimwood B.G. Hechemy K. Stevens R.W. Exp. Parasitol. 1979; 48: 282-286Crossref PubMed Scopus (37) Google Scholar). Extraction and Purification of Glycolipids—Purified tachyzoites were washed twice with phosphate-buffered saline and extracted according to Menon et al. (30Menon A.K. Schwarz R.T. Mayor S. Cross G.A. J. Biol. Chem. 1990; 265: 9033-9042Abstract Full Text PDF PubMed Google Scholar). Briefly, labeled and unlabeled glycolipids were extracted with chloroform/methanol/water (CMW 10:10:3 by volume). CMW-extracted glycolipids were partitioned between water and water-saturated n-butyl alcohol. Glycolipids recovered in the butanol phase were analyzed by TLC on Merck Silica 60 TLC plates using solvent system: hexane/chloroform/methanol/water/acetic acid (3:10:10: 2:1 by volume). Chromatograms were scanned for radioactivity using a Berthold LB 2842 linear analyzer, and areas corresponding to individual glycolipid peaks (I to VI) were scraped off, re-extracted with CMW (10:10:3), and recovered in the butanol phase after water-saturated n-butyl alcohol/water partition. Glycolipids extracted according to the same procedure from non-infected Vero cells were used as control. Generation of Neutral Core Glycans of Glycolipids—TLC-purified glycolipids or chemically synthesized GPIs were dephosphorylated, deaminated, and reduced as described (30Menon A.K. Schwarz R.T. Mayor S. Cross G.A. J. Biol. Chem. 1990; 265: 9033-9042Abstract Full Text PDF PubMed Google Scholar). Briefly, dephosphorylation was performed by incubation of TLC-purified glycolipids with 48% aqueous HF for 60 h at 0 °C. The reaction was stopped by blowing off HF under a nitrogen stream. Glycolipids were resuspended in 400 μl of freshly prepared 0.1 m sodium acetate (pH 3.5) containing 0.25 m NaNO2 and incubated at room temperature for 4 h. The reaction was terminated by addition of 300 μl of 0.4 m boric acid and 130 μl of 1 m NaOH. The material was reduced overnight at 4 °C using 2 m NaBH4 prepared in 0.3 m NaOH and desalted over AG50W-X12 resin. Core glycans were also prepared from the GPI anchor of P30, the major surface protein of T. gondii purified as described by Zinecker et al. (31Zinecker C.F. Striepen B. Geyer H. Geyer R. Dubremetz J.F. Schwarz R.T. Mol. Biochem. Parasitol. 2001; 116: 127-135Crossref PubMed Scopus (29) Google Scholar). Briefly, P30 was isolated from tachyzoite lysates by immunoprecipitation using an affinity column (1E5 monoclonal antibody coupled to CNBr-Sepharose). Eluted P30 was digested with Pronase. Hydrophobic peptides isolated by water-saturated n-butyl alcohol/water partition were dephosphorylated, deaminated, and reduced as described above. Generation of Diacylglycerol and Dialkylglycerol of Glycolipids— TLC-purified GPI of T. gondii and the chemically synthesized GPIb were cleaved using 1 unit of phosphatidylinositol/phospholipase C derived from Bacillus cereus (Sigma) in 100 μl of 0.1 m Tris-HCl (pH 7.4), 0.1% sodium deoxycholate overnight at 37 °C. The reaction was terminated by addition of 10 μl of acetic acid, and the enzyme was inactivated by treatment for 3 min at 100 °C. Diacylglycerol and dialkylglycerol were recovered in the butanol phase after partition between water and water-saturated n-butyl alcohol. In Vitro Macrophage Activation and TNFα Sandwich ELISA—RAW 264.7 macrophages were diluted to a concentration of 106/ml with RPMI 1640 medium supplemented with 5% fetal calf serum, and 0.2-ml aliquots were dispensed into the wells of a 96-well plate. The cells were allowed to adhere for 3 h, washed, and incubated at 37 °C in 5% CO2 atmosphere for 24 h with medium containing different concentrations of GPIs and core glycans purified from T. gondii, or synthesized GPIs. Butanol containing the GPIs and water containing the core glycans were dried under a nitrogen stream and resuspended in culture medium by sonication. For negative controls, macrophages were incubated in medium alone to determine background levels of TNFα production or in medium containing glycolipids purified from non-infected Vero cells. Measurement of TNFα levels in the macrophage culture supernatants was performed using specific sandwich ELISA (BD Biosciences). Samples were incubated at 4 °C overnight in 96-well plate coated with anti-mouse TNFα, followed by washing and probing with biotinylated anti-mouse TNFα, followed by avidin-peroxidase. After addition and development of substrate, the colorimetric reaction was evaluated using automated ELISA reader at 450 nm. Levels of TNFα were calculated from standard curves established for each assay by using mouse recombinant TNFα, and the data from triplicate determinations were expressed as pg/ml (mean ± S.D.). NF-κB p65 Transcription Factor Assay—Macrophages were stimulated for 30 min with or without T. gondii GPI. Total macrophage proteins (20 μg) were analyzed for NF-κB activation, using the Trans-AM™ NF-κB p65 transcription factor assay kit, according to the manufacturer's protocol (Active Motif, Rixensart, Belgium). Statistics—The unpaired Student's t test was adopted for statistical evaluation, and p < 0.05 was considered significant. TNFα Production in Response to GPIs and Their Core Glycans Highly Purified from T. gondii—In order to determine whether the GPIs of T. gondii induce TNFα production, parasite glycolipids were isolated and purified by sequential extraction and phase partition and analyzed by TLC. Glycolipids from metabolically labeled parasites with [3H]glucosamine were used as tracers. The glycolipids have the following structures (32Striepen B. Zinecker C.F. Damm J.B. Melgers P.A. Gerwig G.J. Koolen M. Vliegenthart J.F. Dubremetz J.F. Schwarz R.T. J. Mol. Biol. 1997; 266: 797-813Crossref PubMed Scopus (82) Google Scholar): GPIs I and II, (ethanolamine-PO4)-Manα1–2Manα1–6(Glcα1–4GalNAcβ1–4)Manα1–4GlcNα-inositol-PO4-lipid; GPI III, (ethanolamine-PO4)-Manα1–2Manα1–6(GalNAcβ1–4)Manα1–4GlcNα-inositol-PO4-lipid; GPIs IV and V, Manα1–2Manα1–6(Glcα1–4GalNAcβ1–4)Manα1–4GlcNα-inositol-PO4-lipid; and GPI VI, Manα1–2Manα1–6(GalNAcβ1–4)Manα1–4GlcNα-inositol-PO4-lipid. GPIs I and II and GPIs IV and V differ in their fatty acid composition with palmitic and stearic acids as predominant lipids (Fig. 1, A and B). Areas corresponding to the six peaks were scraped and glycolipids re-extracted from silica using organic solvent as described under “Experimental Procedures.” Macrophages were incubated with individual GPIs, and TNFα secretion in supernatants was measured using a sandwich ELISA. All of six T. gondii GPIs induced significantly the production of TNFα by macrophages (Fig. 2A). The stimulation was effective with glycolipids extracted from 106 parasites (p < 0.01) and increased in a dose-dependent manner. The individual separation of the GPIs by TLC is required to observe the production of TNFα. Indeed, total glycolipids extracted from 108 T. gondii tachyzoites with CMW, as well as hydrophobic glycolipids, recovered after a butanol/water partition, did not induce the secretion of TNFα by macrophages (Fig. 2A). In order to confirm that glycolipids of T. gondii are responsible for this effect, non-infected Vero cells were tested as control. Glycolipids were extracted from 107 Vero cells using CMW and butanol/water partition and separated on TLC. Areas corresponding to the GPIs of T. gondii were scraped and glycolipids re-extracted from silica. The different samples failed to induce the production of TNFα by macrophages (Fig. 2B), confirming that this production is specific for the individually separated GPIs from T. gondii. In order to know if the glycan moiety induces TNFα production, core glycans from individual T. gondii GPIs were prepared by dephosphorylation, nitrous deamination, and reduction. Two core glycans were generated from the six glycolipids; the core glycan A, having the structure Manα1–2Manα1–6(GalNAcβ1–4)Manα1–4-anhydromannitol, and the core glycan B, having the structure Manα1–2Manα1–6(Glcα1–4GalNAcβ1–4)Manα1–4-anhydromannitol. Core glycans were tested for their ability to stimulate macrophages and showed to significantly induce the production of TNFα at a concentration of 100 nm (p < 0.01) (Fig. 2C). Lower concentration (10 nm) had no significant effect in comparison with the control culture. To rule out the possibility that another molecule (impurity) was responsible for TNFα production, a second method was used to obtain highly purified core glycan from T. gondii GPIs. Immunoprecipitation of p30, the major surface GPI-anchored protein of T. gondii, permitted isolation of a pure GPI-anchored protein free of other glycolipids. Purified p30 was digested with Pronase, and hydrophobic anchor peptide was treated to release the neutral core glycan fragment. This highly purified molecule was tested and showed to stimulate TNFα production in macrophages (Fig. 2C). This confirmed that GPIs extracted from T. gondii are able to induce TNFα production in macrophages and that the neutral core glycans are sufficient for this effect.Fig. 2GPIs and their core glycans highly purified from T. gondii tachyzoites induce TNFα production in macrophages. A, macrophages were incubated for 24 h in the presence of glycolipids extracted with CMW (10:10:3) from 108 T. gondii tachyzoites, or in the presence of glycolipids recovered in butanol, or in the presence of GPIs separated by TLC (I–VI) extracted from 106 to 109 T. gondii tachyzoites. B, macrophages were cultured for 24 h in presence of glycolipids extracted from 107 Vero cells and corresponding to the GPIs of T. gondii on the TLC. C, macrophages were incubated for 24 h with 10 nm and 100 nm of core glycans prepared from the GPIs (I–VI) or from the major surface protein p30 of T. gondii. Supernatants were assayed for TNFα production using a sandwich ELISA. Values represent the mean ± S.D. of triplicate samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TNFα Production in Response to Chemically Synthesized GPIs without Lipid Moiety and with Dialkylglycerol as Lipid Moiety—To understand the role of the GPI lipid moiety in activation of macrophages, a T. gondii GPI anchor chemically synthesized without the lipid moiety, GPIa (Manα1–2Manα1–6(GalNAcβ1–4)Manα1–4GlcNα-inositol-PO4, Fig. 3A), was tested. In the presence of GPIa, macrophages secreted TNFα in the supernatant (Fig. 3B). As for core glycans generated from GPIs of T. gondii, the core glycan of the synthetic GPIa induced TNFα production (Fig. 3C). The core glycan of GPIa has the same structure as that of the core glycan of GPI III, and the production of TNFα induced by both core glycans is comparable. Only 10 nm of the GPIa core glycan was sufficient to stimulate significantly the macrophages, whereas 1 μm (100-fold more) of the entire molecule was required for the same effect, suggesting that the phosphate group of GPIa diminishes efficiency of the core glycan. The role of the lipid moiety in TNFα stimulation was further investigated using a second synthetic molecule. GPIb has the same sugar structure as the molecule described above, but it contains dialkylglycerol as lipid moiety (Fig. 4A). No difference in TNFα level was observed between control and cells stimulated with the synthetic GPIb, even at 100 μm (Fig. 4B). Preparation of the core glycan from GPIb resulted in an active molecule that stimulated TNFα production in macrophages (Fig. 4C). In order to determine whether GPIb could inhibit the production of TNFα, the molecule was tested in addition to a natural GPI extracted from T. gondii or with the synthetic GPIa. The GPIb molecule reduced significantly TNFα production induced by high levels of both natural and synthetic GPIs (Fig. 4D). This indicates that the presence of dialkylglycerol as lipid moiety leads to a molecule unable to stimulate TNFα production, which inhibits the active GPIs. T. gondii GPI and the chemically synthesized GPIb were treated with phosphatidylinositol/phospholipase C, and the corresponding lipid moieties were recovered in the butanol phase. This treatment leads to complete release of the lipid moiety (33Striepen B. Tomavo S. Dubremetz J.F. Schwarz R.T. Biochem. Soc. Trans. 1992; 20: 296Crossref PubMed Scopus (11) Google Scholar). The GPI diacylglycerol alone was able to induce TNFα production but to a lesser extent than the entire molecule (Fig. 4E). A dipalmitoylglycerol standard tested as a control did not have any effect, whereas a distearylglycerol standard leads to secretion of TNFα. The released dialkylglycerol, as the intact GPIb, did not induce TNFα production and inhibited the stimulatory effect of the GPI diacylglycerol (Fig. 4E).Fig. 4A chemically synthesized GPI with dialkylglycerol as lipid moiety does not induce TNFα production in macrophages, whereas its core glycan does. A, structure of GPIb, a chemically synthesized GPI anchor of T. gondii with dialkylglycerol as lipid moiety. B, macrophages were cultured for 24 h with 1–100 μm of GPIb. C, macrophages were incubated for 24 h in the presence of 10 and 100 nm of GPIb core glycan. D, macrophages were cultured in presence of a natural GPI extracted from 108 T. gondii tachyzoites (G), or in the presence of 100 μm of GPIa (a), with or without 10 μm GPIb (b). E, macrophages were incubated for 24 h with dialkylglycerol (GPIb) or diacylglycerol (T. gondii GPI), or both together, or with dipalmitoylglycerol (diC16) and distearylglycerol (diC18) as standards. Supernatants were assayed for TNFα production using a sandwich ELISA. Values represent the mean ± S.D. of triplicate samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) NF-κB p65 Activation in Macrophages in Response to T. gondii GPI—The NFκB/c-rel family of transcription factors regulates the expression of a wide range of genes involved in inflammation, including the gene coding for TNFα. To determine whether the production of TNFα in response to the GPI molecules of T. gondii is regulated by NF-κB, macrophages were incubated 15, 30, and 60 min with or without T. gondii GPI. The proteins of macrophages were extracted and tested in an NF-κB assay. After 15 min of incubation, the GPI did not increase NF-κB activation in macrophages in comparison with the control (Fig. 5). After 30 and 60 min, NF-κB activation increased in the presence of T. gondii GPI. Incubation with the synthetic GPIb reduced NF-κB activation induced after 30 min by the natural GPI of T. gondii. This suggests that the TNFα production by macrophages in response to T. gondii GPIs requires NF-κB activation. T. gondii-induced host cell activation is supposed to play a role in the initiation of immune resistance and may regulate intracellular replication of the parasite. Expression and secretion of cytokines triggered by living Toxoplasma or parasite lysate are involved in acute reaction of the T cell-independent immunity (34Sher A. Oswald I.P. Hieny S. Gazzinelli R.T. J. Immunol. 1993; 150: 3982-3989PubMed Google Scholar, 35Gazzinelli R.T. Hieny S. Wynn T.A. Wolf S. Sher A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6115-6119Crossref PubMed Scopus (750) Google Scholar). The bioactive factors and the signaling pathways responsible for this response are not clearly determined. Grunvald et al. (14Grunvald E. Chiaramonte M. Hieny S. Wysocka M. Trinchieri G. Vogel S.N. Gazzinelli R.T. Sher A. Infect. Immun. 1996; 64: 2010-2018Crossref PubMed Google Scholar) have shown a major role for heat-stable protein and carbohydrate structural components in the induction of IL-12, TNFα, IL1β, and IL-10. The authors hypothesize that the relevant structures are protein-anchored glycolipids similar to the phosphatidylinositol-containing molecules in the malaria parasites responsible for the stimulation of TNFα (14Grunvald E. Chiaramonte M. Hieny S. Wysocka M. Trinchieri G. Vogel S.N. Gazzinelli R.T. Sher A. Infect. Immun. 1996; 64: 2010-2018Crossref PubMed Google Scholar). Indeed, GPI of P. falciparum, free or associated with protein, induced TNFα and IL-1 secretion by macrophages (23Schofield L. Hackett F. J. Exp. Med. 1993; 177: 145-153Crossref PubMed Scopus (397) Google Scholar). Similarly, highly purified GPI anchors isolated from trypomastigotes of T. cruzi triggered the synthesis of TNFα by mouse macrophages (26Almeida I.C. Camargo M.M. Procopio D.O. Silva L.S. Mehlert A. Travassos L.R. Gazzinelli R.T. Ferguson M.A. EMBO J. 2000; 19: 1476-1485Crossref PubMed Scopus (200) Google Scholar). Here we show that the GPIs highly purified from T. gondii stimulate macrophages to produce TNFα. Only individually separated GPIs of T. gondii present an activity. Indeed, glycolipids extracted with chloroform/methanol/water or hydrophobic glycolipids recovered in butanol after a butanol/water partition do not induce TNFα production, suggesting the presence of other glycolipids than GPIs that could have an inhibitory effect. Thus, the structure and the role in vivo of inhibitory molecules remain to be elucidated. The malarial GPI core glycan Manα1–2Manα1–6Manα1–4GlcN-myo-inositol is necessary and sufficient to induce TNFα production, whereas the inositol glycan or the lyso-GPI is not sufficient for TNFα induction, indicating that the GPI-associated diacylglycerol is required for maximal downstream gene expression (25Tachado S.D. Gerold P. Schwarz R. Novakovic S. McConville M. Schofield L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4022-4027Crossref PubMed Scopus (184) Google Scholar). Our results show that the two neutral core glycans prepared from free or protein-associated GPIs of T. gondii induce TNFα secretion. Furthermore, the lipid moiety alone is able to stimulate the macrophages. A GPI anchor of T. gondii synthesized with a phosphate group instead of fatty acids is able to induce TNFα secretion but to a lesser extent than the core glycans. On the contrary, a GPI anchor synthesized with a dialkylglycerol as the lipid moiety does not stimulate the production of TNFα in macrophages. Furthermore, this molecule inhibits the secretion of TNFα in response to both natural and synthetic GPIs of T. gondii. Interestingly, the dialkylglycerol moiety alone is sufficient to induce an inhibitory effect. The structurally related molecule iM4 glycoinositol phospholipid from L. mexicana is unable to induce TNFα and inhibits the TNFα production in response to LPS stimulation, suggesting that alkylacylglycerols may antagonize macrophage activation (25Tachado S.D. Gerold P. Schwarz R. Novakovic S. McConville M. Schofield L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4022-4027Crossref PubMed Scopus (184) Google Scholar). Thus, the results suggest that only GPIs with acylglycerols as the lipid moieties are able to induce TNFα synthesis in macrophages. It would be interesting to study if a chemically synthesized GPI of T. gondii with an alkylacylglycerol as the lipid moiety also may inhibit TNFα production. The inhibitory activity of GPIb could be exploited at first in an animal model to treat acute toxoplasmosis, where high levels of TNFα are produced. The inositol glycan moiety of the malarial GPI is the minimal structural requirement for activation of protein-tyrosine kinases in macrophages. However, to induce maximal gene expression, a non-protein-tyrosine kinase signal is required and is provided by the diacylglycerols, which activate the calcium independent ϵ isoform of the protein kinase C (25Tachado S.D. Gerold P. Schwarz R. Novakovic S. McConville M. Schofield L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4022-4027Crossref PubMed Scopus (184) Google Scholar). In the case of T. cruzi, GPIs activate the extracellular signal-related kinases (ERK1/ERK2), the c-Jun kinases, and the stress-activated protein kinase (p38). The activation of these signaling pathways results in changes in gene expression, mediated by transcription factors (CREB and NF-κB), leading to IL-12 and TNFα synthesis (36Ropert C. Ferreira L.R. Campos M.A. Procopio D.O. Travassos L.R. Ferguson M.A. Reis L.F. Teixeira M.M. Almeida I.C. Gazzinelli R.T. Microbes Infect. 2002; 4: 1015-1025Crossref PubMed Scopus (64) Google Scholar). Infection of macrophages with T. gondii induces rapid phosphorylation of ERK1/ERK2 and p38 (19Butcher B.A. Denkers E.Y. Infect. Immun. 2002; 70: 5216-5224Crossref PubMed Scopus (77) Google Scholar). Experiments performed with protein kinase inhibitors suggest that the synthesis of TNFα, induced by soluble antigen of T. gondii, is protein kinase C-dependent, whereas the kinase does not appear to play a dominant role in soluble antigen-induced IL-12 and IL-10 responses, arguing that distinct parasite signals are involved in monokine induction (14Grunvald E. Chiaramonte M. Hieny S. Wysocka M. Trinchieri G. Vogel S.N. Gazzinelli R.T. Sher A. Infect. Immun. 1996; 64: 2010-2018Crossref PubMed Google Scholar). In vivo infection of mice with virulent or avirulent strains of T. gondii leads to activation of NF-κB in peritoneal cells (37Shapira S. Speirs K. Gerstein A. Caamano J. Hunter C.A. J. Infect. Dis. 2002; 185: 66-72Crossref PubMed Scopus (107) Google Scholar). In contrast, Butcher and Denkers (19Butcher B.A. Denkers E.Y. Infect. Immun. 2002; 70: 5216-5224Crossref PubMed Scopus (77) Google Scholar) have no evidence for NF-κB p65 translocation in peritoneal cells of mice infected in vivo with a virulent strain of T. gondii. Despite rapid IκBα degradation, the parasite fails to induce NF-κB nuclear translocation as well as induction of IL-12 and TNFα in macrophages (19Butcher B.A. Denkers E.Y. Infect. Immun. 2002; 70: 5216-5224Crossref PubMed Scopus (77) Google Scholar). In vitro infection with tachyzoites of avirulent strains stimulates the translocation of NF-κB to the nucleus. This translocation is reduced in cells exposed to virulent strains (38Dobbin C.A. Smith N.C. Johnson A.M. J. Immunol. 2002; 169: 958-965Crossref PubMed Scopus (100) Google Scholar). Soluble tachyzoite antigen prepared from T. gondii induces NF-κB DNA binding activity in macrophages (20Gazzinelli R.T. Wysocka M. Hieny S. Scharton-Kersten T. Cheever A. Kuhn R. Muller W. Trinchieri G. Sher A. J. Immunol. 1996; 157: 798-805PubMed Google Scholar). We reported that T. gondii GPI activates NF-κB p65 in macrophages after 30 min of stimulation. The inhibition of TNFα production in the presence of GPI with dialkylglycerol as lipid moiety correlates with a reduction of NF-κB activation, suggesting that GPI-induced TNFα expression is under the regulatory control of the NF-κB p65. A remaining issue concerns the mechanisms by which the GPIs enter the signaling pathway. A possibility is that GPIs are hydrolyzed at the cell surface by GPI-specific phospholipase. Alternatively, the passive incorporation of intact GPI-anchored proteins is a well known phenomenon, as well as mono-molecular, micellar, and micro-vesicular transport (39Medof M.E. Nagarajan S. Tykocinski M.L. FASEB. J. 1996; 10: 574-586Crossref PubMed Scopus (179) Google Scholar). Despite the growing evidence implicating GPIs from parasitic protozoa in the induction of cytokine synthesis by macrophages, not much is known about the receptors that are triggered by these GPIs. Tachado et al. (25Tachado S.D. Gerold P. Schwarz R. Novakovic S. McConville M. Schofield L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4022-4027Crossref PubMed Scopus (184) Google Scholar) suggest expression of a glycan-specific receptor responsible for activation of protein-tyrosine kinases and the translocation of the phosphatidylinositol moiety of the malarial GPI to the cytoplasm to act as a second messenger. Magez et al. (40Magez S. Stijlemans B. Radwanska M. Pays E. Ferguson M.A. De Baetselier P. J. Immunol. 1998; 160: 1949-1956PubMed Google Scholar) evoke the involvement of two distinct receptors for the glycan and lipid components of T. brucei GPIs. Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators (41Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752PubMed Google Scholar, 42Lien E. Sellati T.J. Yoshimura A. Flo T.H. Rawadi G. Finberg R.W. Carroll J.D. Espevik T. Ingalls R.R. Radolf J.D. Golenbock D.T. J. Biol. Chem. 1999; 274: 33419-33425Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar). Results obtained by Campos et al. (43Campos M.A. Almeida I.C. Takeuchi O. Akira S. Valente E.P. Procopio D.O. Travassos L.R. Smith J.A. Golenbock D.T. Gazzinelli R.T. J. Immunol. 2001; 167: 416-423Crossref PubMed Scopus (450) Google Scholar) suggest that although functionally similar, the receptors used by LPS and GPI-mucin of T. brucei are not the same. Indeed, involvement of TLR-2, but not TLR-4, is essential in the induction of IL-12, TNFα, and NO by murine macrophages activated by T. cruzi GPI (43Campos M.A. Almeida I.C. Takeuchi O. Akira S. Valente E.P. Procopio D.O. Travassos L.R. Smith J.A. Golenbock D.T. Gazzinelli R.T. J. Immunol. 2001; 167: 416-423Crossref PubMed Scopus (450) Google Scholar). In human monocytes, both CD14 and TLR-2 are important molecules in the signaling transduction induced by the GPI-mucin of T. cruzi (36Ropert C. Ferreira L.R. Campos M.A. Procopio D.O. Travassos L.R. Ferguson M.A. Reis L.F. Teixeira M.M. Almeida I.C. Gazzinelli R.T. Microbes Infect. 2002; 4: 1015-1025Crossref PubMed Scopus (64) Google Scholar). B cell proliferation in response to T. gondii heat shock protein HSP70 requires expression of TLR-4 (44Aosai F. Chen M. Kang H.K. Mun H.S. Norose K. Piao L.X. Kobayashi M. Takeuchi O. Akira S. Yano A. Cell Stress Chaperones. 2002; 7: 357-364Crossref PubMed Scopus (53) Google Scholar), and the induction of IL-12 by T. gondii soluble antigens is abolished in peritoneal macrophages lacking MyD88, the adaptor molecule used by TLR family members (45Scanga C.A. Aliberti J. Jankovic D. Tilloy F. Bennouna S. Denkers E.Y. Medzhitov R. Sher A. J. Immunol. 2002; 168: 5997-6001Crossref PubMed Scopus (383) Google Scholar). These new findings suggest that a receptor of the Toll-like receptor family could be responsible for signaling in macrophages in response to T. gondii GPIs. In this case, the inhibitory effect of GPI with dialkylglycerol as lipid moiety could be due to a competition for this receptor.