The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum have been proposed to be the major factors that contribute to malaria pathogenesis through their ability to induce proinflammatory responses. In this study we identified the receptors for P. falciparum GPI-induced cell signaling that leads to proinflammatory responses and studied the GPI structure-activity relationship. The data show that GPI signaling is mediated mainly through recognition by TLR2 and to a lesser extent by TLR4. The activity of sn-2-lyso-GPIs is comparable with that of the intact GPIs, whereas the activity of Man3-GPIs is about 80% that of the intact GPIs. The GPIs with three (intact GPIs and Man3-GPIs) and two fatty acids (sn-2-lyso-GPIs) appear to differ considerably in the requirement of the auxiliary receptor, TLR1 or TLR6, for recognition by TLR2. The former are preferentially recognized by TLR2/TLR1, whereas the latter are favored by TLR2/TLR6. However, the signaling pathways initiated by all three GPI types are similar, involving the MyD88-dependent activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 and NF-κB-signaling pathways. The signaling molecules of these pathways differentially contribute to the production of various cytokines and nitric oxide (Zhu, J., Krishnegowda, G., and Gowda, D. C. (2004) J. Biol. Chem. 280, 8617-8627). Our data also show that GPIs are degraded by the macrophage surface phospholipases predominantly into inactive species, indicating that the host can regulate GPI activity at least in part by this mechanism. These results imply that macrophage surface phospholipases play important roles in the GPI-induced innate immune responses and malaria pathogenesis. The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum have been proposed to be the major factors that contribute to malaria pathogenesis through their ability to induce proinflammatory responses. In this study we identified the receptors for P. falciparum GPI-induced cell signaling that leads to proinflammatory responses and studied the GPI structure-activity relationship. The data show that GPI signaling is mediated mainly through recognition by TLR2 and to a lesser extent by TLR4. The activity of sn-2-lyso-GPIs is comparable with that of the intact GPIs, whereas the activity of Man3-GPIs is about 80% that of the intact GPIs. The GPIs with three (intact GPIs and Man3-GPIs) and two fatty acids (sn-2-lyso-GPIs) appear to differ considerably in the requirement of the auxiliary receptor, TLR1 or TLR6, for recognition by TLR2. The former are preferentially recognized by TLR2/TLR1, whereas the latter are favored by TLR2/TLR6. However, the signaling pathways initiated by all three GPI types are similar, involving the MyD88-dependent activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 and NF-κB-signaling pathways. The signaling molecules of these pathways differentially contribute to the production of various cytokines and nitric oxide (Zhu, J., Krishnegowda, G., and Gowda, D. C. (2004) J. Biol. Chem. 280, 8617-8627). Our data also show that GPIs are degraded by the macrophage surface phospholipases predominantly into inactive species, indicating that the host can regulate GPI activity at least in part by this mechanism. These results imply that macrophage surface phospholipases play important roles in the GPI-induced innate immune responses and malaria pathogenesis. Malaria caused by the parasitic protozoa of the genus Plasmodium is a major public health problem in Africa, South America, and South Asia (1Gilles H.M. Warrell D.A. Bruce-Chawatt's Essential Malariology. Arnold Publishers, London1997: 1-340Google Scholar, 2Sherman I.W. Malaria: Parasite Biology, Pathogenesis, and Protection. American Society for Microbiology, Washington, D. C.1998: 1-575Google Scholar, 3Snow R.W. Craig M.H. Deichmann U. Marsh K. Bull. W. H. O. 1999; 77: 624-640PubMed Google Scholar, 4Sachs J. Malaney P. Nature. 2002; 415: 680-685Crossref PubMed Scopus (1411) Google Scholar). The disease afflicts about 500 million people and causes ∼3 million deaths annually and ranks first among the various infectious diseases, causing global morbidity and mortality. More than 100 Plasmodium species exist in nature that can infect various vertebrate animals (1Gilles H.M. Warrell D.A. Bruce-Chawatt's Essential Malariology. Arnold Publishers, London1997: 1-340Google Scholar). However, only four species are infectious to man, and of these, Plasmodium falciparum is responsible for >95% of deaths (1Gilles H.M. Warrell D.A. Bruce-Chawatt's Essential Malariology. Arnold Publishers, London1997: 1-340Google Scholar, 2Sherman I.W. Malaria: Parasite Biology, Pathogenesis, and Protection. American Society for Microbiology, Washington, D. C.1998: 1-575Google Scholar, 3Snow R.W. Craig M.H. Deichmann U. Marsh K. Bull. W. H. O. 1999; 77: 624-640PubMed Google Scholar, 4Sachs J. Malaney P. Nature. 2002; 415: 680-685Crossref PubMed Scopus (1411) Google Scholar). Plasmodium infection causes a wide range of clinical manifestations, including cerebral malaria, acute respiratory distress, pulmonary edema, renal failure, and severe anemia (1Gilles H.M. Warrell D.A. Bruce-Chawatt's Essential Malariology. Arnold Publishers, London1997: 1-340Google Scholar, 2Sherman I.W. Malaria: Parasite Biology, Pathogenesis, and Protection. American Society for Microbiology, Washington, D. C.1998: 1-575Google Scholar). The acquisition of effective protective immunity to malaria requires repetitive infections over a period of a few years (5Baird J.K. Parasitol. Today. 1995; 11: 105-111Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 6Riley E.M. Hviid L. Theander T.G. Kierszenbaum F. Malaria. Academic Press, Inc., New York, NY1994: 119-143Google Scholar). Therefore, during the initial periods of infection, innate immunity plays a crucial role in controlling parasite growth (7Smith T. Felger I. Tanner M. Beck H.P. Trans. R. Soc. Trop. Med. Hyg. 1999; 93: 59-64Abstract Full Text PDF PubMed Scopus (160) Google Scholar, 8Stevenson M.M. Riley E.M. Nat. Rev. 2004; 4: 169-180Crossref Scopus (0) Google Scholar, 9Kaufmann S.H.E. Sher A. Ahmed R. Immununology of Infectious Diseases. American Society for Microbiology, Washington, D. C.2002: 1-495Google Scholar). Otherwise, the parasite is expected to grow exponentially, leading to the rapid destruction of all the circulatory erythrocytes and death. Proinflammatory cytokines such as TNF-α, 1The abbreviations used are: TNF, tumor necrosis factor; IL, interleukin; GPI, glycosylphosphatidylinositol; PI, phosphatidylinositol; TLR, toll-like receptor; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; NF-κB, nuclear factor κB; MyD88, myeloid differentiation factor; LPS, lipopolysaccharide; MALP-2, macrophage activating lipoprotein, 2 kDa; Pam3CSK4, N-palmitoyl-S-dipalmitoyl-cysteinyl-SKKKK (tripalmytoyl-CSK4 peptide); PE, phycoerythrin; FACS, fluorescence-activated cell sorter; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; HPLC, high performance liquid chromatography; HPTLC, high performance TLC; ELISA, enzyme-linked immunosorbent assay. interferon-γ, IL-12, IL-1, and IL-6, and nitric oxide produced during malaria infection are critical for controlling parasite growth (2Sherman I.W. Malaria: Parasite Biology, Pathogenesis, and Protection. American Society for Microbiology, Washington, D. C.1998: 1-575Google Scholar, 8Stevenson M.M. Riley E.M. Nat. Rev. 2004; 4: 169-180Crossref Scopus (0) Google Scholar, 9Kaufmann S.H.E. Sher A. Ahmed R. Immununology of Infectious Diseases. American Society for Microbiology, Washington, D. C.2002: 1-495Google Scholar, 10Biron C.A. Gazzinelli R.T. Curr. Opin. Immunol. 1995; 7: 485-497Crossref PubMed Scopus (218) Google Scholar, 11Clark I.A. Hunt N.H. Butcher G.A. Cowden W.B. J. Immunol. 1987; 139: 3493-3496PubMed Google Scholar, 12van der Heyde H.C. Pepper B. Batchelder J. Cigel F. Weidanz W.P. Exp. Parasitol. 1997; 85: 206-213Crossref PubMed Scopus (89) Google Scholar, 13Su Z. Stevenson M.M. Infect. Immun. 2000; 68: 4399-4406Crossref PubMed Scopus (200) Google Scholar, 14Su Z. Stevenson M.M. J. Immunol. 2002; 168: 1348-1355Crossref PubMed Scopus (145) Google Scholar, 15Balmer P. Phillips H.M. Maestre A.E. McMonagle F.A. Phillips R.S. Parasite Immunol. 2000; 22: 97-106Crossref PubMed Scopus (40) Google Scholar). However, excessive production of proinflammatory cytokines could lead to severe pathological conditions (16Playfair J.H.L. Taverne J. Bate C.A. de Souza J.B. Immunol. Today. 1990; 11: 25-27Abstract Full Text PDF PubMed Scopus (2) Google Scholar, 17Hommel M. Ann. Trop. Med. Parasitol. 1993; 87: 627-635Crossref PubMed Scopus (32) Google Scholar, 18Grau G.E. De Kossodo S. Parasitol. Today. 1994; 10: 408-409Abstract Full Text PDF PubMed Scopus (78) Google Scholar, 19Kwiatkowski D. Parasitol. Today. 1995; 11: 206-212Abstract Full Text PDF PubMed Scopus (91) Google Scholar). Therefore, understanding the mechanism of innate immune responses to P. falciparum factors could offer therapeutic targets for malaria. Although the various P. falciparum components that are potentially involved in the production of inflammatory responses by the innate immune system remain to be elucidated, the glycosylphosphatidylinositol (GPI) anchor glycolipids of the parasite have been proposed as the prominent parasite components responsible for malaria pathogenesis (20Schofield L. Hackett F. J. Exp. Med. 1993; 177: 145-153Crossref PubMed Scopus (406) Google Scholar, 21Schofield L. Vivas L. Hackett F. Gerold P. Schwarz R.T. Tachado S. Ann. Trop. Med. Parasitol. 1993; 87: 617-626Crossref PubMed Scopus (89) Google Scholar). Accumulated evidence indicates that GPIs of parasitic protozoa contribute prominently to the pathology of parasitic diseases (22Ropert C. Gazzinelli T. Curr. Opin. Microbiol. 2000; 3: 395-403Crossref PubMed Scopus (85) Google Scholar). The deleterious effects of the parasite GPIs have been attributed to their ability to induce TNF-α and other proinflammatory cytokines and nitric oxide, which contribute to disease pathology (20Schofield L. Hackett F. J. Exp. Med. 1993; 177: 145-153Crossref PubMed Scopus (406) Google Scholar, 21Schofield L. Vivas L. Hackett F. Gerold P. Schwarz R.T. Tachado S. Ann. Trop. Med. Parasitol. 1993; 87: 617-626Crossref PubMed Scopus (89) Google Scholar, 22Ropert C. Gazzinelli T. Curr. Opin. Microbiol. 2000; 3: 395-403Crossref PubMed Scopus (85) Google Scholar). It has been shown that the level of TNF-α is markedly elevated in patients with fatal cerebral malaria, and anti-TNF-α antibodies prevent the development of cerebral malaria (23Kwiatkowski D. Hill A.V. Sambou I. Twumasi P. Castracane J. Manogue K.R. Cerami A. Brewster D.R. Greenwood B.M. Lancet. 1990; 336: 1201-1204Abstract PubMed Scopus (780) Google Scholar). GPIs consist of a conserved glycan structure, ethanolamine-phosphate-6Manα1-2Manα1-6Manα1-4GlcN, α(1-6)-linked to the PI (24Englund P.T. Annu. Rev. Biochem. 1993; 62: 121-138Crossref PubMed Google Scholar, 25McConville M.J. Ferguson M.A.J. Biochem. J. 1993; 294: 305-324Crossref PubMed Scopus (805) Google Scholar, 26Ferguson M.A.J. Brimacombe J.S. Brown J.R. Crossman A. Dix A. Field R.A. Gu ̈ther M.L. Milne K.G. Sharma D.K. Smith T.K. Biochim. Biophys. Acta. 1999; 1455: 327-340Crossref PubMed Scopus (125) Google Scholar, 27Ferguson M.A.J. J. Cell Sci. 1999; 112: 2799-2809Crossref PubMed Google Scholar). GPIs are ubiquitous in eukaryotes, where they are primarily involved in anchoring certain cell surface proteins to plasma membranes. Compared with animal cells, GPIs are abundantly expressed in parasites, such as Trypanosoma, Leishmania, and Plasmodium. Therefore, in these parasites a relatively large pool of GPIs is no longer anchored to proteins and appears to be the direct target of the host innate immune system for inducing proinflammatory responses (22Ropert C. Gazzinelli T. Curr. Opin. Microbiol. 2000; 3: 395-403Crossref PubMed Scopus (85) Google Scholar). GPIs from different species differ in the type of acyl/alkyl substituents, the presence of additional sugar moieties on the third and/or first mannose, extra ethanolamine phosphate groups on the carbohydrate moiety, and acyl substituent on C2 of inositol, leading to a broad structural diversity and variation in potency of their biological activity (28Almeida I.G. Gazzinelli T.T. J. Leukocyte Biol. 2001; 70: 467-477PubMed Google Scholar, 29Gowda D.C. Microbes Infect. 2002; 4: 983-990Crossref PubMed Scopus (26) Google Scholar). GPIs of P. falciparum have been shown to activate protein-tyrosine kinase and protein kinase C, which together regulate the activation of NF-κB/c-Rel transcription factor with the downstream expression of proinflammatory responses (30Schofield L. Novakovic S. Gerold P. Schwarz R.T. McConville M.J. Tachado S.D. J. Immunol. 1996; 156: 1886-1896PubMed Google Scholar, 31Tachado S.D. Gerold P. McConville M.J. Baldwin T. Quilici D. Schwarz R.T. Schofield L. J. Immunol. 1996; 156: 1897-1907PubMed Google Scholar, 32Tachado 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 (185) Google Scholar). However, the receptors that mediate P. falciparum GPI signaling and how the exogenously induced signal is transmitted into the cells has remained unclear. Research during the past few years has shown that the innate immune responses to various microbial pathogens are mediated by a family of signal-transducing proteins called TLRs (33Takeda K. kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4772) Google Scholar, 34Akira S. Curr. Opin. Immunol. 2003; 15: 5-11Crossref PubMed Scopus (478) Google Scholar, 35Takeda K. Akira S. J. Dermatol. Sci. 2004; 34: 73-82Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). To date, 13 TLRs, TLR1 through TLR13, have been identified in mammalian cells, most recognizing specific pathogen-associated molecular patterns (36Tabeta K. Georgel P. Janssen E. Du X. Hoebe K. Crozat K. Mudd S. Shamel L. Sovath S. Goode J. Alexopoulou L. Flavell R.A. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3516-3521Crossref PubMed Scopus (805) Google Scholar). For example, TLR4 recognizes enterobacterial LPS, TLR3 recognizes double-stranded RNA, TLR5 recognizes flagellin, and TLR9 recognizes CpG-containing motifs of bacterial DNA. TLR2, however, exhibits broad ligand recognition, and the identified ligands include peptidoglycan, lipoteichoic acid, lipoproteins, lipoarabinomannan, zymosan, certain glycolipids, non-enterobacterial LPS, and porins (33Takeda K. kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4772) Google Scholar, 34Akira S. Curr. Opin. Immunol. 2003; 15: 5-11Crossref PubMed Scopus (478) Google Scholar, 35Takeda K. Akira S. J. Dermatol. Sci. 2004; 34: 73-82Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Efficient signal transduction by TLR2 appears to require its heterodimerization with either TLR1 (for triacylated lipoproteins) or TLR6 (for diacylated lipoproteins) (33Takeda K. kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4772) Google Scholar, 37Takeda K. Takeuchi O. Akira S. J. Endotoxin Res. 2002; 8: 459-463Crossref PubMed Scopus (191) Google Scholar, 38Akira S. Hemmi H. Immunol. Lett. 2003; 85: 85-95Crossref PubMed Scopus (930) Google Scholar). The transmission of responses by TLRs involves in most cases the recruitment of a shared adaptor protein, MyD88, which interacts with TLRs through Toll-IL-1 receptor (TIR) domains, initiating signaling cascades, engaging various MAPKs and NF-κB (39Barton G.M. Madzhitov R. Science. 2003; 300: 1524-1525Crossref PubMed Scopus (1062) Google Scholar). Thus far there have been no direct studies demonstrating TLR-mediated immune responses to P. falciparum ligands, although MyD88-deficient mice have been reported to be protected from Plasmodium berghei-induced IL-12-mediated liver injury, suggesting involvement of TLR-mediated immune responses to malarial factors (40Adachi K. Tsutsui H. Kashiwamura S. Seki E. Nakano H. Takeuchi O. Takeda K. Okumura K. Van Kaer L. Okamura H. Akira S. Nakanishi K. J. Immunol. 2001; 167: 5928-5934Crossref PubMed Scopus (191) Google Scholar). Recently, GPI moieties of the mucin-type glycoproteins of Trypanosoma cruzi trypamastigotes have been shown to induce proinflammatory responses through TLR2-mediated signaling (41Ropert C. Almeida I.C. Closel M. Travassos L.R. Ferguson M.A. Cohen P. Gazzinelli R.T. J. Immunol. 2001; 166: 3423-3431Crossref PubMed Scopus (101) Google Scholar, 42Campos 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 (459) Google Scholar, 43Campos M.A. Closel M. Valente E.P. Cardoso J.E. Akira S. Alvarez-Leite J.I. Ropert C. Gazzinelli R.T. J. Immunol. 2004; 172: 1711-1718Crossref PubMed Scopus (151) Google Scholar). However, P. falciparum GPIs are structurally distinct from those of T. cruzi; the former contain a diacylated glycerol moiety and fatty acid acylation at C-2 of inositol, whereas the latter have sn-1-alkyl-sn-2-acylglycerol and lack inositol acylation (29Gowda D.C. Microbes Infect. 2002; 4: 983-990Crossref PubMed Scopus (26) Google Scholar, 44Gerold P. Dieckmann-Schuppert A. Schwarz R.T. J. Biol. Chem. 1994; 269: 2597-2606Abstract Full Text PDF PubMed Google Scholar, 45Naik R.S. Branch O.H. Woods A.S. Vijaykumar M. Perkins D.J. Nahlen B.L. Lal A.A. Cotter R.J. Costello C.E. Ockenhouse C.F. Davidson E.A. Gowda D.C. J. Exp. Med. 2000; 192: 1563-1575Crossref PubMed Scopus (199) Google Scholar). Furthermore, the requirement of TLR1 or TLR6 for heterodimerization with TLR2 for signaling by GPIs is not known. In this study we show that proinflammatory responses to P. falciparum GPIs by macrophages are mediated mainly through TLR2 and to a lesser but significant extent also through TLR4. We also show for the first time that the parasite GPIs are degraded by macrophage surface phospholipase A2 and phospholipase D and that intact and sn-2-lyso-GPIs are differentially recognized by TLR2/TLR1 and TLR2/TLR6. Materials—All cell culture reagents, including RPMI 1640, DMEM, FBS, penicillin/streptomycin, and gentamycin were from Invitrogen. In some experiments FBS from Hyclone (Logan, UT) was used. Bee venom phospholipase A2 (1724 units/mg), jack bean α-mannosidase (20 units/mg), LPS (from Salmonella minnesota Re595 strain, catalog number L 9764) were from Sigma. MALP-2 and Pam3CSK4 (standard TLR2 ligands) were from EMC Microcollections (Tübingen, Germany). IL-1β was from Pierce. Human blood and serum from healthy donors were from the hospital of the Hershey Medical Center. Limulus amebocyte lysate assay kit (catalog number GS003 with sensitivity of 0.03 enzyme units/ml) was from Associates of Cape Cod (Falmouth, MA). Mycoplasma detection kit (version 2.0) was from American Type Culture Collection (ATCC). Colloidal gold (40-60-nm particle size) was from ImmunoReagent products (Lakeside, AZ). Chemiluminescence substrate kit was from KPL (Gaithersburg, MD). Dual luciferase reporter assay kit and passive lysis buffer were from Promega (Madison, WI). The Golgi Stop (monensin), 2.4 G2-purified antibody against Fc receptor, Cytofix/Cytoperm, and PE-conjugated rat anti-mouse CD11b (clone M1/70) were from Pharmingen, and fluorescein isothiocyanate-conjugated rat anti-mouse TNF-α (clone MP6-XT22) was from Caltag (Burlingame, CA). Anti-human TLR2 and anti-human TLR4 mouse monoclonal antibodies (both IgG2), clones TL2.1 and HTA125, respectively, were from eBioscience, Inc. (San Diego, CA). A mouse monoclonal antibody (IgG2) specific to an ovarian glycoprotein tumor antigen (OVB-3, Ref. 46Willingham M.C. FritGerald D.J. Pastan I. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 15916-15922Crossref Scopus (54) Google Scholar) was a gift from Dr. Ira Pastan, NCI, NIH, Bethesda, MD. Phosphospecific anti-mouse ERK1/ERK2, p38, and JNK mouse monoclonal antibodies, anti-mouse β-tubulin peptide mouse monoclonal antibody, rabbit polyclonal antibodies against mouse IκBα, ERK1/ERK2, p38, and JNK peptides, and horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were from Cell Signaling Technology, Inc. (Beverly, MA). Mouse monoclonal antibody-specific HA (HA.11 from clone 16B12) was from Covance, Richmond, CA. Nitrocellulose membranes were from Bio-Rad. Endotoxin-free reagents, water, and buffers were used for all the experimental procedures. Cell Lines—Raw264.7 and J774A.1 mouse macrophage cell lines, and L929 murine fibroblast cells were from ATCC. HEK-293 human embryonic kidney epithelial cells were originally from Dr. David Schowalter, when he was at the University of Washington. HEK-293, Raw264.7, and J774A.1 cells were cultured in DMEM, 10% FBS, 1% penicillin/streptomycin. L929 cells were cultured in DMEM, 5% FBS, 1% glutamine, and 1% penicillin/streptomycin in roller flasks at 37 °C. For the preparation of conditioned medium from L929 cells, the cells were cultured in the above medium for 5 days, and the supernatant was collected and centrifuged at 2500 rpm for 20 min. The clear solution was used as a source of macrophage colony stimulatory factor. Mice—The TLR2-/-, TLR4-/-, and MyD88-/- mice were produced at the Research Institute for Microbial Diseases, Osaka University, Japan. The knock-out mice were backcrossed six to eight generations to C57BL/6J mice. The C57BL/6J wild type mice were from The Jackson Laboratories. TLR2 and TLR4 double knock-out (TLR2/4-/-) mice were produced by crossing TLR2-/- and TLR4-/- mice. All animals were maintained in a pathogen-free environment. Parasites—Intraerythrocytic P. falciparum (FCR-3 strain) was cultured in RPMI 1640 medium using O-type blood and 10% O-positive human serum, 50 μg/ml gentamycin at 3-4% hematocrit (45Naik R.S. Branch O.H. Woods A.S. Vijaykumar M. Perkins D.J. Nahlen B.L. Lal A.A. Cotter R.J. Costello C.E. Ockenhouse C.F. Davidson E.A. Gowda D.C. J. Exp. Med. 2000; 192: 1563-1575Crossref PubMed Scopus (199) Google Scholar, 47Naik R.S. Davidson E.A. Gowda D.C. J. Biol. Chem. 2000; 275: 24506-24511Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Parasite cultures were regularly synchronized with 5% sorbitol (48Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2855) Google Scholar) and tested for mycoplasma by PCR using an ATCC kit (49Rowe J.A. Scrag I.G. Kwiatkowski D.J.P. Ferguson D.J. Carucci D.J. Newbold C.I. Mol. Biochem. Parasitol. 1998; 92: 177-180Crossref PubMed Scopus (40) Google Scholar). Isolation of GPIs from P. falciparum—Isolation of parasites and purification of GPIs were performed as described previously (45Naik R.S. Branch O.H. Woods A.S. Vijaykumar M. Perkins D.J. Nahlen B.L. Lal A.A. Cotter R.J. Costello C.E. Ockenhouse C.F. Davidson E.A. Gowda D.C. J. Exp. Med. 2000; 192: 1563-1575Crossref PubMed Scopus (199) Google Scholar). Briefly, mycoplasma-free parasite cultures with 20-30% parasitemia were harvested at the schizont stage, treated with 0.025% saponin in Trager buffer (10 mm K2HPO4, 1 mm NaH2PO4, 11 mm NaHCO3, 56 mm NaCl, 59 mm KCl, 14 mm glucose, pH 7.4; Ref. 45Naik R.S. Branch O.H. Woods A.S. Vijaykumar M. Perkins D.J. Nahlen B.L. Lal A.A. Cotter R.J. Costello C.E. Ockenhouse C.F. Davidson E.A. Gowda D.C. J. Exp. Med. 2000; 192: 1563-1575Crossref PubMed Scopus (199) Google Scholar), and passed through a 26-gauge needle to lyse the erythrocytes. The suspension was centrifuged and washed several times, and the erythrocyte debris was removed by centrifugation on a 5% bovine serum albumin cushion. The parasites were washed 3 times with phosphate-buffered saline, pH 7.4, lyophilized, and stored at -80 °C. The parasites (from 10 ml wet pellet) were extracted 3 times with chloroform, methanol (2:1, v/v) to remove non-glycosylated lipids. GPIs were extracted with chloroform, methanol, water (10:10:3, v/v/v), dried, and partitioned between water and water-saturated 1-butanol. The organic layer was washed four times with water and dried. The residue was extracted with 80% aqueous 1-propanol and dried and the GPIs were further purified by HPLC using C4 Supelcosil LC-304 column (4.6 × 250 cm, Supelco) as described previously (45). In some experiments GPIs were further purified by HPTLC using chloroform, methanol, water (10:10:2.4, v/v/v). The HPLC and HPTLC-purified GPIs were found to be free from endotoxin as tested by the Limulus amebocyte lysate assay (50Sakai H. Hisamoto S. Fukutomi I. Sou K. Takeoka S. Tsuchida E. J. Pharm. Sci. 2004; 93: 310-321Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). By this assay (with positive detection limit of 0.01 ng/ml for LPS standard), 5 μg/ml GPIs showed negative endotoxin activity. The purity of the GPIs was confirmed by mass spectrometry and carbohydrate compositional analysis. The GPIs were quantified by determining the amount of GlcN and Man after acid hydrolysis and HPLC as described previously (45Naik R.S. Branch O.H. Woods A.S. Vijaykumar M. Perkins D.J. Nahlen B.L. Lal A.A. Cotter R.J. Costello C.E. Ockenhouse C.F. Davidson E.A. Gowda D.C. J. Exp. Med. 2000; 192: 1563-1575Crossref PubMed Scopus (199) Google Scholar). Preparation of Man3-GPIs—The Man4-GPIs (10 μg) were treated with jack bean α-mannosidase (40 units/ml) in 100 μl of 100 mm sodium acetate, 2 mm Zn2+, pH 5.0, containing 0.1% sodium taurodeoxycholate at 37 °C for 24 h (51Milne K.G. Ferguson M.A.J. Englund P.T. J. Biol. Chem. 1999; 274: 1465-1471Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The solutions were heated in a boiling water bath for 5 min, cooled, and extracted with water-saturated 1-butanol, washed 4 times with water, and dried. The Man3-GPIs were purified by HPLC as above, and the purity of the samples was ascertained by mass spectrometry and by the carbohydrate compositional analysis. Preparation of sn-2-lyso-GPIs—The GPIs (10 μg) were treated with bee venom phospholipase A2 (1700 unit/ml) in 100 μl of 100 mm Tris-HCl, 10 mm CaCl2, pH 7.5, at 37 °C for 24 h (44Gerold P. Dieckmann-Schuppert A. Schwarz R.T. J. Biol. Chem. 1994; 269: 2597-2606Abstract Full Text PDF PubMed Google Scholar). The solution was heated in a boiling water bath for 5 min, cooled, and extracted with water-saturated 1-butanol, washed 4 times with water, and dried. The sn-2-lyso-GPIs were purified by HPLC as above, and the purity was determined by mass spectrometry and by carbohydrate compositional analysis (45Naik R.S. Branch O.H. Woods A.S. Vijaykumar M. Perkins D.J. Nahlen B.L. Lal A.A. Cotter R.J. Costello C.E. Ockenhouse C.F. Davidson E.A. Gowda D.C. J. Exp. Med. 2000; 192: 1563-1575Crossref PubMed Scopus (199) Google Scholar). Mass Spectrometry—The solutions of GPIs in chloroform, methanol, and water (8:4:3, v/v/v) were mixed with equal volumes of a saturated solution of 6-aza-2-thiothymine in 50% ethanol, deposited on the sample plate, and air-dried. Mass spectra (an average of 50 shots) were acquired in linear negative ion mode on a DE-PRO matrix-assisted laser desorption ionization time-of-flight mass spectrometer (PE-Biosystems, Framingham, MA) equipped with a nitrogen laser (337 nm) at 20-kV accelerating voltage. Carbohydrate Compositional Analysis—The GPIs (∼1 μg each) were hydrolyzed with 400 μl of either 2.5 m trifluoroacetic acid at 100 °C for 5 h or 3 m HCl at 100 °C for 4 h. The hydolysates were dried in a Speed-Vac, dissolved in water, and analyzed on a CarboPac PA10 high pH anion-exchange column (2 × 250 mm) using a Dionex BioLC GS50 HPLC system coupled to a ID50 electrochemical detector (52Hardy M.R. Townsend R.R. Methods Enzymol. 1994; 230: 208-225Crossref PubMed Scopus (157) Google Scholar). The elution was performed with 16 mm sodium hydroxide, and the response factors for the monosaccharides were determined using standard sugar solutions. Coating of GPIs to Gold Particles—The colloidal gold suspension (1.5 ml) was centrifuged in an Eppendorf centrifuge at 8000 rpm and washed 3 times with endotoxin-free water. The particle pellet thus obtained (∼8 μl) was suspended in 120 μl of water and mixed with GPIs (5 μg) in 30 μl of 80% 1-propanol and dried in a Speed-Vac. By adding a known amount of [3H]GlcN-labeled GPIs during coating, we found that the GPIs are quantitatively adsorbed by the gold particles. The GPI-coated gold particles were suspended in 13 ml of DMEM, 10% FBS and used for stimulation of macrophages in 24- or 96-well microtiter plates. Preparation of Mouse Bone Marrow Macrophages and Human Peripheral Blood Monocytes and Stimulation with GPIs—Mouse macrophages were obtained by the differentiation of primary bone marrow cells with 30% of L929 cell-conditioned medium as described (53Zhu J. Krishnegowda G. Gowda D.C. J. Biol. Chem. 2005; 280: 8617-8627Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The macrophages were plated into 96-well plates (2.5 × 104 cells/well), and after 24 h, the culture supernatants were removed and incubated with the indicated amounts of GPIs in DMEM medium containing 10% FBS and 1% penicillin/streptomycin. For human monocytes, the whole blood was diluted with 3 volumes of RPMI 1640 medium, and 4 volumes of cell suspension was layered on 1 volume of isolymph and centrifuged at 1300 rpm at room temperature for 30 min. The buffy layer at the interface was recovered, washed 2 times with RPMI 1640 medium, and then suspended in RPMI 1640 medium containing 10% FBS (Invitrogen) and 1% penicillin/streptomycin (2.5 × 1