Requirement of Mitogen-Activated Protein Kinases and IB Phosphorylation for Induction of Proinflammatory Cytokines Synthesis by Macrophages Indicates Functional Similarity of Receptors Triggered by Glycosylphosphatidylinositol Anchors from Parasitic Protozoa and Bacterial Lipopolysaccharide 1 Catherine Ropert,* Igor C. Almeida, ² Meire Closel,* Luiz R. Travassos, ‡ Michael A. J. Ferguson, § Philip Cohen, ¶ and Ricardo T. Gazzinelli 2 * In the present study, we evaluated the ability of GPI-anchored mucin-like glycoproteins purified from Trypanosoma cruzi trypo- mastigotes (tGPI-mucin) to trigger phosphorylation of different mitogen-activated protein kinases (MAPKs) and related tran- scription factors in inflammatory macrophages. Kinetic experiments show that the peak of extracellular signal-related kinase (ERK)-1/ERK-2, stress-activated protein kinase (SAPK) kinase-1/mitogen-activated protein kinase (MAPK) kinase-4, and p38/ SAPK-2, phosphorylation occurs between 15 and 30 min after macrophage stimulation with tGPI-mucin or GPI anchors highly purified from tGPI-mucins (tGPI). The use of the specific inhibitors of ERK-1/ERK-2 (PD 98059) and p38/SAPK-2 (SB 203580) phosphorylation also indicates the role of MAPKs, with possible involvement of cAMP response element binding protein, in triggering TNF- and IL-12 synthesis by IFN--primed-macrophages exposed to tGPI or tGPI-mucin. In addition, tGPI-mucin and tGPI were able to induce phosphorylation of IB, and the use of SN50 peptide, an inhibitor of NF-B translocation, resulted in 70% of TNF- synthesis by macrophages exposed to tGPI-mucin. Finally, the similarity of patterns of MAPK and IB phosphorylation, the concentration of drugs required to inhibit cytokine synthesis, as well as cross-tolerization exhibited by macrophages exposed to tGPI, tGPI-mucin, or bacterial LPS, suggest that receptors with the same functional properties are triggered by these different microbial glycoconjugates. The Journal of Immunology, 2001, 166: 3423–3431. T he cellular compartment from the innate immune system has low levels of specificity and can be activated imme- diately after infection through the involvement of cell re- ceptors specific for dominant structures (e.g., LPS, lipopeptides, lipoteichoic acid, repetitive mannose structures, and DNA CpG motifs) that are unique and characteristic molecules from certain groups of specific pathogens (1–7). Cells of the macrophage lin- eage exposed to such microbial components will synthesize high levels of proinflammatory cytokines that induce multiple activities in other cells of the immune system. Notably, cells from macro- phage lineage exposed to protozoan parasites produce IL-12 and TNF- that are responsible for initiating IFN- synthesis by NK cells (8–10). In agreement, different studies indicate that during the early stages of infection, before the establishment of acquired protective immunity, the cellular compartment of the innate im- mune system plays a crucial role in host resistance against different intracellular protozoa (8 –10). To better understand the early stimulation of the innate immune system by parasitic protozoa, studies performed in our laboratories and elsewhere have focused on the identification and chemical characterization of the protozoan products that trigger the proin- flammatory and effector functions of macrophages. These studies indicate that tGPI, 3 a GPI anchor purified from the mucin-like glycoprotein (tGPI-mucin) of Trypanosoma cruzi trypomastigotes, has an essential role in triggering various macrophage functions (11–16), similar to the importance of LPS in infection with Gram- negative bacteria. Comparable results were obtained with the GPI anchors purified from Plasmodium falciparum and Trypanosoma brucei (Refs. 17 and 18; see review in Ref. 19). Recent studies have suggested that protozoan GPI anchors may have two signaling portions (i.e., the glycan core and inositolphos- pholipid) that trigger different signaling components responsible for cytokine and NO synthesis by mammalian host cells (20 –23). *Rene ´ Rachou Research Center-Fundac ¸ao Oswaldo Cruz and Department of Bio- chemistry and Immunology, Instituto de Cie ˆncias Biolo ´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; ² Department of Parasitology, Instituto de Cie ˆncias Biolo ´gicas, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Brazil; ‡ Discipline of Cell Biology Biologia Celular, Universidade Federal de Sa ˜o Paulo, Sa ˜o Paulo, Brazil; and § Division of Molecular Parasitology and ¶ Medical Research Council Protein Phosphorylation Unit, Department of Biochemistry, University of Dundee, Dundee, United Kingdom Received for publication June 13, 2000. Accepted for publication December 21, 2000. 1 This work was supported in part by the World Health Organization (Grant A00477), Conselho Nacional de Desenvolvimento de Pesquisas Cientı ´fica e Tecnolo ´gica (Grants 522.056/95-4 and 521.117/98), and Fundac ¸a ˜o de Amparo a Pesquisa do Es- tado de Minas Gerais (CBB-2343/98). C.R. is a visiting scientist, and I.C.A., L.R.T. and R.T.G. are research fellows from Conselho Nacional de Desenvolvimento de Pesquisas Cientı ´fica e Tecnolo ´gica. I.C.A. is supported by Grant 98/10495-5 from Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado de Sa ˜o Paulo. 2 Address correspondence and reprint requests to Dr. Ricardo T. Gazzinelli, Labora- tory of Immunopathology, Centro de Pesquisas Rene ´ Rachou, Fundac ¸ao Oswaldo Crus, Av. Augusto de Lima 1715, Barro Preto, 30190-002, Belo Horizonte, MG, Brazil. E-mail address: ritoga@dedalus.lcc.ufmg.br 3 Abbreviations used in this paper: tGPI, GPI anchor purified from tGPI-mucin; tGPI- mucin, GPI-anchored mucin-like glycoproteins derived from Trypanosoma cruzi try- pomastigotes; CREB, cAMP response element binding protein; ATF, activating tran- scription factor; ERK, extracellular signal-related kinase; MAPK, mitogen-activated protein kinase; JNK, c-jun N-terminal kinase; iNOS, inducible NO synthase; SAPK, stress-activated protein kinase; SKK, SAPK kinase; MKK, MAPK kinase; TLR, Toll- like receptor. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00