Abstract
Introduction Bacterial infection results in activation of the innate im-mune system. The recognition of a pathogen is mediated bya set of germline-encoded receptors that are referred to aspattern-recognition receptors (PRRs) that can directly rec-ognize invariant molecular structures known as pathogen-associated molecular patterns (PAMPs), which are sharedby large groups of microorganisms. One PAMP recognizedby PRRs is lipopolysaccharide (LPS), a mayor componentof the outer membrane of Gram-negative bacteria. LPSstimulates host cells and makes them produce various proin-flammatory cytokines, such as TNF- a , IL-1 a , and IL-6.LPS is captured by LPS-binding protein (LBP), which de-livers to CD14 on the cell surface. Because CD14 lacks anintracellular signaling domain, a transmembrane coreceptorhas been postulated in LPS-responsive cells that transducesthe LPS signaling into the cytoplasm. In Drosophila , theToll receptor participates in establishing the dorsoventralpatterns in the embryo and inducing antifungal immune re-sponse in the adult fly. Toll is a type I transmembrane pro-tein with a cytoplasmic domain with sequence homology tothe interleukin-1 receptor (IL-1R). Activation of Toll leadsto induction of genes through the transcription factor NF-kBpathway (1). Recently, several Toll-like receptors (TLRs)have been identified in macrophages and monocytes, basedon homology to Drosophila protein. Other studies havevariably suggested that TLR-2 and TLR-4 serve as the mainmediators of responses to LPS (2). In addition, TLR-2 mayalso act as a signaling receptor for other common bacterialstructures: peptidoglycan (PGN) and lipoteichoic acid(LTA) from Gram-positive bacteria and lipoarabinomannan(LAM) from Mycobacterium. Lipophosphopeptidoglycan (LPPG) antigens from Ent-amoeba histolytica were described for the first time by Isi-basi et al., and were later used to study its expression underdifferent culture conditions and in different E. histolytica strains using monoclonal antibodies. One antibody againstLPPG was able to inhibit adhesion of amebas and cytotoxic-ity to target cells. Recently, the expression of LPPG wascorrelated to amebic virulence. In addition, the protectionagainst invasive amebiasis by a single monoclonal antibodydirected against LPPG has been studied (3). We studiedLPPG, and found that, due to its polysaccharide nature, thisameba surface molecule behaves like a PAMP. LPPG isprobably recognized by TLR-2, but instead of inducing apro-inflammatory response, it induced production of inter-leukin-10 and downregulation of tlr -2 gene expression. Materials and Methods Isolation of LPPG. Trophozoites from E. histolytica strainHM-1 were cultivated under axenic conditions, and wereused to extract LPPG using the phenol–water method de-scribed by Westphal and Jann and modified by Isibasi et al.Contaminating LPS in LPPG preparations were determinedusing quantitative Limulus lysate assay (BioWhittaker). Inall preparations, 0.020 ng of LPS/ m g of carbohydrate wasdetected. Monocyte isolation and LPPG/LPS stimulation assay. Periph-eral blood mononuclear cells were isolated on Ficoll-paquegradients (Pharmacia LKB Biotecnology) and cultured (5 3 10
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