Glycosylphosphatidylinositol (GPI) anchors and glycoinositolphospholipids (GIPLs) from parasitic protozoa have been shown to exert a wide variety of effects on cells of the host innate immune system. However, the receptor(s) that are triggered by these protozoan glycolipids has not been identified. Here we present evidence that Trypanosoma cruzi-derived GPI anchors and GIPLs trigger CD25 expression on Chinese hamster ovary-K1 cells transfected with CD14 and Toll-like receptor-2 (TLR-2), but not wild-type (TLR-2-deficient) Chinese hamster ovary cells. The protozoan-derived GPI anchors and GIPLs containing alkylacylglycerol and saturated fatty acid chains or ceramide were found to be active in a concentration range of 100 nM to 1 μM. More importantly, the GPI anchors purified from T. cruzi trypomastigotes, which contain a longer glycan core and unsaturated fatty acids in the sn-2 position of the alkylacylglycerolipid component, triggered TLR-2 at subnanomolar concentrations. We performed experiments with macrophages from TLR-2 knockout and TLR-4 knockout mice, and found that TLR-2 expression appears to be essential for induction of IL-12, TNF-α, and NO by GPI anchors derived from T. cruzi trypomastigotes. Thus, highly purified GPI anchors from T. cruzi parasites are potent activators of TLR-2 from both mouse and human origin. The activation of TLR-2 may initiate host innate defense mechanisms and inflammatory response during protozoan infection, and may provide new strategies for immune intervention during protozoan infections.

Vespa et al., 1994;Biron and Gazzinelli, 1995 Aliberti et al., 1996;Fearon and Locksley, 1996; Trinchieri and Scott, 1996). In addition, the early Seder et al., 1993; Abbas et al., 1996; Fearon macrophage proinflammatory cytokines by Trypanoand Locksley, 1996). During infection with T.cruzi, the soma cruzi is considered to be important in controlling induction of parasite-specific CMI is likely to be involved the infection and the outcome of Chagas' disease. Here in at least two aspects of Chagas' disease pathophysiology we show that the potent tumour necrosis factor-α-, (Vespa et al., 1994;Fearon and Locksley, 1996; Brener interleukin-12-and nitric oxide-inducing activities of and Gazzinelli, 1997). The first is the control of parasite T.cruzi trypomastigote mucins were recovered quantireplication and its spread in the vertebrate host tissues. tatively in a highly purified and characterized glycosyl-The second is the inflammatory reaction observed in the phosphatidylinositol (GPI) anchor fraction of this infected host tissues, which is likely to be a major cause material. The bioactive trypomastigote GPI fraction of cardiac tissue damage during the acute and chronic was compared with a relatively inactive GPI fraction phases of the disease. prepared from T.cruzi epimastigote mucins. The trypoRelatively little is known about the protozoan parasite mastigote GPI structures were found to contain addimolecules that initiate the synthesis of pro-inflammatory tional galactose residues and unsaturated, instead of cytokines and nitric oxide (NO) by macrophages. Recent saturated, fatty acids in the sn-2 position of the alkylstudies have suggested a role for glycosylphosphatidylacylglycerolipid component. The latter feature is esseninositol (GPI) anchors from Plasmodium falciparum tial for the extreme potency of the trypomastigote (Schofield and Hackett, 1993;Schofield et al., 1996; GPI fraction, which is at least as active as bacterial Tachado et al., 1996Tachado et al., , 1997 and Trypanosoma brucei endotoxin and Mycoplasma lipopeptide and, therefore, (Magez et al., 1998) in this process. However, no evidence one of the most potent microbial proinflammatory of the biochemical purity of the P.falciparum GPIs was agents known.provided, making estimates of their concentrations dubiKeywords: Chagas' disease/cytokines/glycosylphosphaous. Furthermore, the possibility of Mycoplasma lipopeptidylinositol/inflammation/nitric oxide tide (Mühlradt et al., 1997) contamination (that has confounded other studies into proinflammatory factors) cannot be formally excluded. In the case of the T.brucei GPI work, highly purified and structurally characterized

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