Leishmania lipophosphoglycan (LPG) activates NK cells through toll-like receptor-2

Molecular & Biochemical Parasitology 130 (2003) 65–74

Leishmania lipophosphoglycan (LPG) activates NK cells through toll-like receptor-2夽 Ingeborg Becker a,∗ , Norma Salaiza a , Magdalena Aguirre a , José Delgado a , Nuria Carrillo-Carrasco a , Laila Gutiérrez Kobeh a , Adriana Ruiz a , Rocely Cervantes a , Armando Pérez Torres b , Nallely Cabrera c , Augusto González a , Carmen Maldonado d , Armando Isibasi d a

d

Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México, Dr. Balmis 148, Colonia Doctores, 06726 Mexico D.F., Mexico b Departamento de Biolog´ıa Celular y Tisular, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico D.F., Mexico c Departamento de Bioqu´ımica, Instituto de Fisiolog´ıa Celular, Universidad Nacional Autónoma de México, Mexico D.F., Mexico Unidad de Investigación Médica en Inmunoqu´ımica, Hospital de Especialidades, Centro Médico Nacional Siglo XXI, IMSS, Mexico D.F., Mexico Received 3 February 2003; received in revised form 10 June 2003; accepted 12 June 2003

Abstract Toll-like receptors (TLRs) mediate the cellular response to conserved molecular patterns shared by microorganisms. We report that TLR-2 on human NK cells is upregulated and stimulated by Leishmania major lipophosphoglycan (LPG), a phosphoglycan belonging to a family of unique Leishmania glycoconjugates. We found that purified L. major LPG upregulates both mRNA and the membrane expression of TLR-2 in NK cells. Additionally, IFN-␥ and TNF-␣ production and nuclear translocation of NF-␬B was enhanced. The activation effect was more intense with LPG purified from infectious metacyclic parasites than from noninfectious procyclic Leishmania. Since the difference between the molecules derived from these two stages of the parasite growth cycle lies exclusively in the number of phosphosaccharide repeat domains and in the composition of glycan side chains that branch off these domains, we propose that TLR-2 possibly distinguishes between phosphorylated glycan repeats on LPG molecules. The effect of LPG on cytokine production and on membrane expression of TLR-2 could be blocked with F(ab )2 fragments of the mAb against LPG (WIC 79.3). Confocal microscopy demonstrated the co-localization of LPG and TLR-2 on the NK cell membrane. Binding of LPG to TLR-2 in NK cells was demonstrated by immunoprecipitations done with anti-TLR-2 and anti-LPG mAb followed by immunoblotting with anti-LPG and anti-TLR-2, respectively. Both antibodies recognized the immune complexes. These results suggest that NK cells are capable of recognition of, and activation by, Leishmania LPG through TLR-2, enabling them to participate autonomously in the innate immune system and thereby increasing the effective destruction of the parasite. © 2003 Elsevier B.V. All rights reserved. Keywords: Leishmania major; Innate immunity; TLR-2; Cytokines

1. Introduction Abbreviations: ADCC, antibody dependent cell cytotoxicity; GIPL, glycoinositol phospholipid; iNOS, inducible nitric oxide synthase; LPG, lipophosphoglycan; MCP-1, macrophage chemoattractant protein 1; MIP1␣, macrophage inflammatory protein 1␣; NF-␬B, nuclear factor ␬B; PAMP, pathogen-associated molecular pattern; PI, phosphatidyl inositol; PMA, phorbol 12-myristate 13-acetate; PNA, peanut agglutinin; TLR, toll-like receptor 夽 This work was supported by grants IN-222599 and IN231602-3 from DGAPA-UNAM and 37538-M from CONACyT to I.B. 夽 Corresponding author. Tel.: +52-55-56232674; fax: +52-55-57610249. E-mail address: [email protected] (I. Becker). 0166-6851/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0166-6851(03)00160-9

Toll-like receptors (TLRs) regulate mammalian innate immune responses identifying “pathogen-associated molecular patterns” (PAMPs) such as glycolipids, peptidoglycans and lipopeptides, shared by large groups of microorganisms [1]. TLRs have been described on many types of cells including blood leukocytes, where recognition of PAMPs leads to the production of pro-inflammatory cytokines, antimicrobial compounds and co-stimulatory molecules in the host cells [2]. Recently, GPI anchors and glycoinositol phospholipids (GIPLs) from Trypanosoma cruzi were reported to activate

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TLR-2, leading to macrophage production of IL-12, TNF-␣ and NO [3]. The early recognition of pathogens by cells capable of synthesizing IL-12 and TNF-␣ is crucial for the adequate control of intracellular pathogens such as Leishmania major. Leishmania cell surfaces are dominated by GPI-anchored or GPI-related molecules as well as abundant GIPLs that are not attached to a protein and that tend to form dense layers on the parasite surface, above which other GPI-anchored molecules, such as lipophosphoglycan (LPG), project [4]. LPG, a large and complex glycophospholipid, is one of the major surface molecules of Leishmania parasite and consists of a type-2 GPI membrane anchor (Man␣1-3Man␣1-4GlcN␣1-6PI motif) that is attached to a long phosphosaccharide-repeat domain, which carries species-specific side-chain modifications and is capped by a neutral oligosaccharide [4–6]. The salient feature of LPG is the repeating phosphorylated saccharide region containing multiple units of a backbone structure of PO4 -6Gal(␤1,4)Man(␣1) attached via a phosphosaccharide core to a 1-O-alkyl-2-lyso-phosphatidyl(myo)inositol anchor. In L. major, the phosphosaccharide core contains the unusual sugar galactofuranose, which is extremely unusual in eukaryotic glycoconjugates [7]. One of the noteworthy features of the backbone is the 4-O-substituted mannose residue, which is not present in any other known eukaryotic glycoconjugate [8]. LPG expression undergoes developmentally regulated, stage-specific biochemical and morphological changes as the parasite differentiates from a noninfective avirulent procyclic promastigote to the infective virulent metacyclic form. During this differentiation process of metacyclogenesis, which occurs in the sand fly vector, there is an important elongation of LPG with an approximate doubling of the phosphosaccharide repeat domains in addition to changes in the composition of the side chains [9]. There is no counterpart to LPG in any other eukaryote, suggesting that LPG represents an adaptation of Leishmania to its complex life cycle in the sandfly vector and the mammalian host. Due to its structural characteristics and its GPI anchor, LPG could also represent a Leishmania ligand for TLR-2. Leishmania are obligate intracellular parasites of macrophages and epidermal Langerhans cells [10] in the mammalian host, and one of the main cytokines that has been implicated in the disease outcome is IFN-␥, through stimulation of oxidative and nitric oxide pathways. In human and experimental leishmaniasis, it has been shown that NK cells represent an early source of IFN-␥, which is a crucial mediator of innate resistance against this parasite [11–14]. Yet it has been reported, that Leishmania inhibits the synthesis of IL-12 in macrophages, thereby limiting NK cell activation. Thus, it is of interest to analyze if Leishmania LPG can have a direct effect on NK-cell activation. NK cells are involved in immune surveillance against exogenous (e.g. viruses) and endogenous (e.g. tumor cells) aggression, and induce lysis of autologous cells by natural

cytotoxicity as well as by antibody dependent cell cytotoxicity (ADCC). In addition, NK cells elaborate cytokines such as IFN-␥ and TNF-␣, which are involved in the elimination of intracellular pathogens [15–17]. It has previously been shown, that the interaction between NK cells with Leishmania leads to the activation of NK cells with IFN-␥ production and increased cytotoxic activity [18–20]. In the present study, we report the presence of TLR-2 on human NK cells and analyze the interaction of this receptor with Leishmania LPG. We found that LPG purified from metacyclic and procyclic promastigotes of L. major stimulates human NK cells, leading to nuclear translocation of NF-␬B, an increased production of IFN-␥ and TNF-␣, as well as an upregulation of TLR-2 expression. The activation of NK cells was higher with the infective metacyclic LPG than with the noninfective procylic form. This activation could be inhibited with F(ab )2 fragments of a monoclonal antibody against L. major LPG.

2. Materials and methods 2.1. LPG purification L. major promastigotes strain MHOM/SU/73/5-ASKH (a generous gift from Dr. H. Moll, University of Würzburg, Germany) were grown in blood agar (NNN medium) overlaid with Schneider’s Drosophila medium (Life Technologies) supplemented with 10% heat-inactivated FBS at 28 ◦ C. Parasites were subcultured every 3–4 days and grown to a density of 1 × 107 /ml. All parasites used in this study were taken from stationary phase cultures before harvesting for extraction of LPG. Metacyclogenesis was determined with peanut agglutinin (PNA) as described by Sacks et al. [21], incubating 2–5 × 108 promastigotes with 100 ␮g/ml PNA for 1 h at 25 ◦ C, after which the parasites were centrifuged at 150 × g for 5 min and the nonagglutinated parasites in the supernatant were used for the metacyclic LPG extraction, whereas procyclic LPG was extracted from the agglutinated parasites. For LPG purification, 1010 promastigotes were harvested and LPG was extracted and purified as described by McConville et al. [22] with some modifications. Briefly, the supernatant was removed and the pellet was extracted with chloroform/methanol/water (1:2:0.5; v/v) for 2 h at room temperature. The insoluble material was used for LPG extraction with 9% 1-butanol in water (2× 500 ␮l) and the pooled supernatants were vacuum dried. LPG was purified from this fraction by octyl-sepharose chromatography in HPLC using a 1-propanol gradient (5–60%) in 0.1 M ammonium acetate. The preparations were negative for the presence of endotoxin using the Limulus sp. amebocyte lysate assay (E-Toxate Kit; Sigma). Polymyxin B (5 ␮g/ml) was also used to confirm the absence of contaminating LPS. A sample was analyzed for protein contaminants by SDS–PAGE with silver staining. The preparation was devoid of protein contaminants.

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2.2. Purification of NK cells Human NK cells were purified from fresh peripheral blood using Ficoll-Hypaque (Sigma) density gradient centrifugation at 300 × g for 20 min at 20 ◦ C. Cells were suspended in pyrogen-free and sterile RPMI-1640 medium (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, 10 mM HEPES buffer, 100 ␮g/ml penicillin, 160 ␮g/ml gentamicin, 17 mM NaHCO3 . PBMC were adhered for 24 h and nonadherent cells were removed, washed in PBS and incubated with monoclonal antibodies for NK purification by MACS Microbeads (NK cell isolation kit by immunomagnetic cell sorting, Miltenyi Biotec; Bergisch Gladbach, Germany) for 25 min and purified by magnetic sorting. NK purification was also done by cell sorting with flow cytometry using monoclonal antibodies (mAbs) anti-CD3 FITC and anti-CD16/CD56 PE (Coulter Immunotech), in an EPICS Elite ESP flow cytometer (Coulter, Marseille, France). Cells were 98.5% pure. 2.3. Isolation of RNA and RT-PCR of TLR-2 For RT-PCR analysis of TLR-2 mRNA, 1 × 106 NK cells of five different normal blood donors were incubated with 10 ␮g/ml purified metacyclic or procyclic LPG in 1 ml RPMI-1640 medium supplemented with 10% FBS for 24 h at 37 ◦ C, 5% CO2 . Supernatants were collected for cytokine quantitation and NK cells were frozen in TRIzol (Life Technologies). Total RNA was extracted from 1 × 106 NK cells with TRIzol and amplified with Super Script One-Step RT-PCR with Platinum Taq (Life Technologies). The cDNA products were PCR amplified with the TLR-2-specific primer pair 5 -GCC AAA GTC TTG ATT GAT TGG-3 and 5 -TTG AAG TTC TCC AGC TCC TG-3 , selected on the basis of the published human TLR-2 sequence [23]. The TLR-2 RT-PCR fragments were purified and sequenced to confirm the identity of the fragments. PCR was done on the Perkin Elmer Gene Amp PCR System 2400 (Roche, NJ). Automatic sequencing of the PCR product was done in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Densitometric analysis was performed by recording the intensity of the bands with a Multi Image Analyzer (Alpha Innotech Corporation). ␤-Actin was used as an internal standard. An intergroup comparison of the densitometric values was performed by Mann–Whitney U-test. P-values below 0.05 were regarded as statistically significant. 2.4. Anti-LPG mAbs and F(ab )2 fragments The hybridoma for mAbs against L. major LPG (WIC 79.3) was a generous gift from Dr. E. Handman (Walter and Eliza Hall Institute of Medical Research, Australia).

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The hybridoma was grown in RPMI-1640 medium supplemented with 10% heat-inactivated FBS at 37 ◦ C with 5% CO2 . Monoclonal antibodies were obtained from culture supernatant as well as from ascites of inoculated BALB/c mice. The antibodies were purified by sepharose protein-A affinity chromatography. F(ab )2 fragments of the mAb were obtained through pepsin cleavage with 5 ␮g of pepsin for each milligram of antibody and incubated for 24 h at 37 ◦ C. Complete digestion was confirmed with SDS–PAGE. 2.5. Flow cytometry analysis of cell-surface TLR-2 NK cells (1 × 106 ) from five different donors were incubated with 10 ␮g purified procyclic or metacyclic LPG for 24 h in RPMI-1640 medium supplemented with 10% FBS and were washed with PBS. Additionally, 10 ␮g/ml procyclic or metacyclic LPG were preincubated with 239 ␮g/ml F(ab )2 of anti-LPG mAb (WIC 79.3) for 1 h, before incubating the LPG with NK cells for 24 h. The NK cells were fixed with 2% paraformaldehyde, washed and incubated with goat anti-human TLR-2 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) or an isotype-matched control. They were then washed and incubated with FITC-labeled rabbit anti-goat IgG (Zymed, San Francisco, CA). NK cell surface staining was performed by using anti-human mAbs for NK cells: anti-CD56 PE (Becton Dickinson, San Jose, CA). Ten thousand NK cells were acquired for each sample and dead cells were gated out based on their light scattering properties. The expression of TLR-2 was examined by flow cytometry (FACSort BD Immunocytometry Systems, San Jose, CA). Analyses were performed by using CellQuest software (BD Immunocytometry Systems). 2.6. IFN-γ and TNF-α ELISA The effect that L. major metacyclic promastigotes had on the production of IFN-␥ and TNF-␣ by NK cells was analyzed as follows: NK cells were co-incubated with L. major metacyclic promastigotes in a 1:10 relationship in 2000 ␮l RPMI-1640 medium supplemented with 10% FBS for 3 h at 28 ◦ C and then for 21 h at 37 ◦ C, 5% CO2 . Otherwise, 1×106 NK cells were incubated with 10 ␮g/ml LPG purified from procyclic or metacyclic L. major promastigotes in 1 ml RPMI-1640 medium supplemented with 10% heat-inactivated FBS for 24 h at 37 ◦ C, 5% CO2 . For control studies, 10 ␮g/ml procyclic or metacyclic LPG was pre-incubated with 239 ␮g/ml F(ab )2 fragments of anti-LPG mAb (WIC 79.3) for 1 h at room temperature before adding the LPG to the NK cells for a 24 h incubation. Cell-free culture supernatants were harvested and the concentrations of IFN-␥ and TNF-␣ were determined by standard sandwich ELISA. In brief, 96-well microtiter plates (Costar, Corning, NY) were coated with an unconjugated anti-TNF-␣ capture Ab (clone MAb1, 6 ␮g/ml; BD PharMingen, San Diego, CA) or anti-IFN-␥ capture Ab (clone NIB42, 6 ␮g/ml; BD PharMingen) in 100 mM Na2 HPO4 , pH 9.0 for 12 h at 4 ◦ C,

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and blocked with PBS containing 0.05% Tween 20 and 10% FBS. Cell supernatants and recombinant hTNF-␣ standard (BD PharMingen) or recombinant hIFN-␥ standard (R&D Systems) were incubated in RPMI-1640 medium supplemented with 10% FBS for 2 h at room temperature. Bound hTNF-␣ or hIFN-␥ were detected using a biotinylated mouse anti-hTNF-␣ Ab (clone MAb11, 2 ␮g/ml; BD PharMingen), or anti-hIFN-␥ Ab (clone 4S.B3, 2 ␮g/ml; BD Pharmingen) in 1% BSA for 1 h. The plate was developed using streptavidin alkaline phosphatase conjugate (Life Technologies) with p-nitrophenyl phosphate (4 mg/ml, Life Technologies) as substrate. The absorbance at 405 nm was read using a microtiter plate reader, and the concentrations of TNF-␣ and IFN-␥ were calculated from a standard curve of recombinant human TNF-␣ and IFN-␥. The TNF-␣ and IFN-␥ concentration of each sample was calculated by regression analysis using the mean absorbance (average of triplicate readings of the sample added). The detection limit of this assay was ∼15 pg/ml. For the cytokine production analysis, an intergroup comparison was performed by Mann–Whitney U-test. P-values below 0.05 were regarded as statistically significant. 2.7. Nuclear extract preparation and Western blot analysis 7 × 106 NK cells were incubated with 10 ␮g/ml purified procyclic or metacyclic LPG or 10 ng/ml PMA in 3.5 ml RPMI-1640 medium for 1 h at 37 ◦ C, 5% CO2 . Nuclear extracts were obtained as described by Santana et al. [24]. Cells were washed with PBS and lysed by incubating them for 10 min at 4 ◦ C in a detergent-free hypotonic buffer (10 mM Tris, pH 7.6, 10 mM NaCl, 1.5 mM MgCl2 , 0.5 mM EDTA, 1 mM dithiothreitol, 1.5 ␮g/ml leupeptin, and 0.7 mM phenylmethylsulfonyl fluoride). Intact nuclei were washed with this lysis buffer, and nuclear extracts were obtained by incubating the nuclei in extraction buffer (20 mM Tris, pH 8.0, 450 mM KCl, 0.5 mM EDTA, 1 mM dithiothreitol, 1.5 ␮g/ml leupeptin, 5 mM spermidine, and 25% glycerol) for 45 min under constant mild agitation at 4 ◦ C. DNA pellets were eliminated by centrifugation for 15 min at 13,500 × g and protein content of the nuclear extracts was determined by Bradford assay [25]. Equivalent amounts of protein were resolved by 12% SDS–PAGE in Tris/glycine/SDS buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) and blotted onto Immobilon-P transfer membranes (100 V, 1.5 h, 4 ◦ C), membranes were washed three times in TBS-T (20 mM Tris–HCl, 150 mM NaCl, 0.005% Tween 20) and blocked with 3% albumin TBS-T 1 h RT. Membranes were immunoblotted with a polyclonal rabbit anti-NF-␬B antibody (Santa Cruz Biotechnology; dilution 1/200) in albumin and TBS-T. Following four 10 min washings with TBS-T, the membrane was incubated with secondary HRP-conjugated goat anti-rabbit IgG (Biomeda; dilution 1/7000) and washed five times in TBS-T, and bands were detected using enhanced chemiluminescence (SuperSignal

West Pico Chemiluminescent Substrate, Pierce, Rockford, IL), according to manufacturer’s description. Densitometric analysis was performed recording the intensity of the bands with a Multi Image Analyzer (Alpha Innotech Corporation). Unstimulated NK cells were used to normalize all measurements. 2.8. Confocal microscopy Co-localization of TLR-2 and LPG binding sites on NK cells was analyzed by confocal microscopy. NK cells were washed three times in washing buffer (PBS pH 7.4 with 1% bovine serum albumin, 1% normal human AB serum and 0.02 M sodium azide) and incubated with anti-TLR-2 (goat polyclonal IgG, Santa Cruz, CA) at a 1:50 dilution for 1 h at room temperature. Primary Ab was detected with FITC-conjugated rabbit anti-goat IgG 1:50 (Zymed) for 30 min at room temperature. After thorough washing, these cells were incubated with purified L. major LPG (10 ␮g/ml) for 3 h in RPMI-1640 at room temperature, washed and incubated with anti-LPG monoclonal antibody (WIC 79.3, 239 ␮g/ml) for 1 h at room temperature. After washing, they were incubated with biotinilated goat anti-mouse IgG 1:50 (Zymed) for 30 min followed by incubation with streptavidin–rhodamine 1:50 (Zymed) for 30 min. All incubations with antibodies were done in incubation buffer (PBS 7.4, 5% bovine serum albumin, 5% inactivated normal human AB serum and 0.02 M sodium azide). Cells were fixed in 1% paraformaldehyde in PBS. Double-labeled NK cells were analyzed through an inverted MRC 4000 Bio-rad confocal microscope. Images were collected with a 40× oil-immersion objective lens, and were simultaneously obtained in two channels. In the merged image, a yellow color display was interpreted as co-localization of TLR-2 and LPG binding sites on NK cells. 2.9. Immunoprecipitation The binding of LPG to TLR-2 was analyzed by immunoprecipitations of the LPG-TLR-2 complex with the mAb against LPG (WIC 79.3) followed by Western blotting with anti-TLR-2 antibody. The immunoprecipitation was repeated inverting the antibodies. In this case, the precipitation was done with anti-TLR-2 followed by Western blotting with anti-LPG mAb. In both cases, 7 × 106 NK cells were incubated with 10 ␮g/ml metacyclic LPG in RPMI for 1 h at 37 ◦ C, 5% CO2 and the same number of NK cells were incubated in RPMI alone. Cells were washed and lysed in 250 ␮l modified radioimmunoprecipitation (RIPA) buffer (10 mM Tris–HCl, pH 7.4, 1% NP-40, 1 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 10 mM Na3 VO4 , 10 mM NaF, 1 mM dithiothreitol). Cell lysates were spun at 14,000 × g for 10 min at 4 ◦ C. Supernatants from activated and nonactivated NK cells were precleared with protein G-agarose beads (Life Technologies) for 2 h under constant agitation

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at 4 ◦ C and centrifuged at 14,000 × g for 10 min at 4 ◦ C. Protein contents in the supernatants were adjusted to 83 ␮g and immunoprecipitated with 100 ␮g/ml of the mAb against LPG overnight at 4 ◦ C with vortex shaking. For the immunoprecipitation with anti-TLR-2, proteins were adjusted to 320 ␮g and precipitated with 20 ␮l of the antibody. The immunocomplexes were captured with protein G-agarose beads for 2 h on ice under constant mild agitation. Beads were washed six times in cold washing buffer (0.05 M Tris, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% NP-40) and immunoprecipitated proteins were diluted into 2× reducing Laemmli Buffer buffer pH 6.8 (4 ml 10% SDS; 2.5 ml 0.5 M Tris–HCl, 0.4% (w/v) SDS; 1 ml 2-mercaptoethanol and 2 ml glycerol), boiled and run on 10% SDS–PAGE. 2.10. Immunoblotting Proteins were transferred to Immobilon-P membranes (Millipore, Medford, MA). Membranes were blocked with 3% BSA in Tris-buffer saline with 0.05% Tween 20 (TBS-T; 10 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Tween 20). The immunoprecipitation with anti-LPG mAb was followed by incubation with polyclonal goat anti-TLR-2 antibody (Santa Cruz Biotechnology; dilution 1/400 in TBS-T) overnight at 4 ◦ C. After eight washings with TBS-T, the membranes were incubated with secondary HRP-conjugated rabbit anti-goat IgG (Biomeda; dilution 1/5000), and proteins were visualized by chemiluminescence. The immunoprecipitation with anti-TLR-2 was probed with anti-LPG mAb (100 ␮g/ml), followed by incubation with secondary HRP-conjugated goat anti-mouse IgG (Zymed; dilution 1/5000). Immunoblotting of purified uncomplexed LPG was done with 10 ␮g metacyclic LPG which was transferred to nitrocellulose paper, blocked in 5% milk in PBS and probed with anti-LPG mAb (100 ␮g/ml) followed by incubation with secondary HRP-conjugated goat anti-mouse IgG (Zymed; dilution 1/5000). For the immunoblotting of TLR-2, 15 ␮g of an NK cell lysate was subjected to a 10% SDS–PAGE, transferred to Immobilon-P membranes and blotted with anti-TLR-2 antibodies (1/400) followed by secondary HRP-conjugated rabbit anti-goat IgG (1/5000). 3. Results TLR-2 was confirmed on purified human NK cells by sequence analysis of the PCR product obtained with specific TLR-2 primers, revealing 100% identity with the human TLR-2 sequence reported in GenBank (Accession number U88878). 3.1. LPG increases TLR-2 mRNA Incubation of human NK cells with 10 ␮g/ml metacyclic or procyclic LPG for 24 h resulted in an increase of TLR-2

Fig. 1. Upregulation of TLR-2 mRNA in NK cells in response to LPG. Purified NK cells were incubated with 10 ␮g/ml metacyclic or procyclic LPG for 24 h and mRNA for TLR-2 was analyzed by RT-PCR. PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide. The relative expression of TLR-2 mRNA of unstimulated NK cells is shown on lane 1. The stimulation of NK cells with metacyclic and procyclic LPG are shown on lanes 2 and 3, respectively. Values in the histogram represent mean ± standard deviation of five independent experiments. An original representative RT-PCR is shown in the inset.

mRNA expression. The incubation of NK cells with metacyclic LPG increased their mRNA expression of TLR-2 by an average of 42% (P < 0.049) over unstimulated NK cells. The incubation of NK cells with procyclic LPG did not induce any significant increase in the TLR-2 mRNA expression, since the average increase was only 8.5% (P < 0.19) (Fig. 1). 3.2. LPG upregulates the cell-surface expression of TLR-2 The LPG-induced upregulation of the TLR-2 expression in NK cells was analyzed by flow cytometry. As shown in Fig. 2, unstimulated NK cells exhibited a low expression of TLR-2 on the cell surface. This was upregulated in response to a stimulation wit 10 ␮g/ml metacyclic LPG, which led to a 1.73 (±0.47)-fold increase in membrane TLR-2 expression, whereas the stimulation with procyclic LPG induced a 1.26 (±0.25)-fold increase. The specificity of the stimulation of LPG was analyzed using the F(ab )2 fragments of the monoclonal antibody WIC 79.3 which recognizes the phosphorylated repeats of L. major LPG. The use of F(ab )2 fragments avoided binding of the mAb to the Fc␥ RIII(CD16) on NK cells, which could possibly have led to unspecific stimulation. The preincubation of LPG with F(ab )2 fragments of the mAb against LPG diminished the

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stimulatory effect of metacyclic LPG by 22.5%, whereas the F(ab )2 fragments had no inhibitory effect on the procyclic LPG stimulation. The isotype-matched control Ab was negative. Staining with anti-CD3 mAb was also negative (data not shown). 3.3. L. major parasites and purified LPG induce IFN-γ and TNF-α production in NK cells

Fig. 2. Flow cytometric analysis of Leishmania major LPG regulation of TLR-2 membrane expression in NK cells. The NK cells were incubated with 10 ␮g purified procyclic or metacyclic LPG for 24 h. Additionally, 10 ug procyclic or metacyclic LPG were preincubated with 239 ␮g F(ab )2 fragments of WIC 79.3 for 1 h, before incubating the LPG with NK cells for 24 h. Panel A shows the stimulatory effect of metacylic LPG on the membrane expression of TLR-2 in NK cells as compared to unstimulated NK cells. The F(ab )2 fragments reduce the stimulatory effect of metacyclic LPG. Isotype-matched antibodies were used as controls. Panel B shows the effect on membrane TLR-2 expression induced by procyclic LPG. Panel C shows the relative increase in expression of TLR-2, which was calculated by dividing the median fluorescence from stimulated cells by the median fluorescence from unstimulated NK cells. Values in the histogram represent mean ± standard deviation of five independent experiments. The values of unstimulated NK cells (lane 1) were normalized, and the relative increase following stimulation with metacyclic LPG is shown in lane 2. The preincubation of metacyclic LPG with F(ab )2 of WIC 79.3 is shown on lane 3. The stimulation of NK cells with procyclic LPG, and the effect of the preincubation of procyclic LPG with F(ab )2 of WIC 79.3 are shown in lanes 4 and 5, respectively.

The cytokine analysis revealed that L. major parasites exerted a stimulatory effect on cytokine secretion by NK cells after 24 h of coincubation, leading to a significant increase in their IFN-␥ and TNF-␣ production. The IFN-␥ production in NK cells increased from a mean 18.44 to 70.88 pg/ml, whereas the TNF-␣ production increased from a mean 9 to 69 pg/ml. The stimulatory effect of cytokine production seen with intact parasites was also observed when NK cells were incubated with 10 ␮g/ml LPG purified from either metacyclic or procyclic L. major promastigotes. Metacyclic LPG exerted a higher stimulation effect on NK cells than procyclic LPG, since it led to a significant increase in IFN-␥ production from an average 19.71 to 115.25 pg/ml. The incubation of NK cells with procyclic LPG only led to an increase in IFN-␥ production from an average 19.71 to 61.21 pg/ml. Procyclic and metacyclic LPG also significantly increased the TNF-␣ production by NK cells. The incubation of NK cells with metacyclic LPG led to an increase from an average 9.91 to 41.77 pg/ml, whereas procyclic LPG led to an increase from 9.91 to 43.75 pg/ml. The mean stimulation factors are shown in Table 1. The preincubation of both forms of LPG with 239 ␮g/ml of the F(ab )2 fragments of the mAb against LPG (WIC 79.3) for 1 h prior to their incubation with NK cells, reduced their stimulatory effect on cytokine production by NK cells. The mAb significantly inhibited the stimulatory effect of metacyclic LPG on NK cells, reducing the IFN-␥ production from 115.25 to 12.66 pg/ml (P < 0.0027) and the TNF-␣ production from 41.77 to 11.40 pg/ml (P < 0.038). The preincubation of procyclic LPG with the mAb reduced IFN-␥ production from 61.15 to 20.55 pg/ml (P < 0.296) and the TNF-␣ production from 43.75 to 6.5 pg/ml (P < 0.105).

Table 1 Mean stimulation factor of cytokine production by NK cells after incubation with L. major parasites or with purified metacyclic or procyclic LPG L. major

Metacyclic LPG

Procyclic LPG

INF-␥

(9)a

3.8 P < 0.0091

(12)a

5.8 P < 0.0017

3.1 (8)a P < 0.07

TNF-␣

7.7 (5)a P < 0.0209

4.2 (9)a P < 0.0062

4.4 (4)a P < 0.021

NK cells were incubated with L. major parasites, with 10 ␮g/ml metacyclic or procyclic LPG for 24 h. IFN-␥ and TNF-␣ production was measured in culture supernatants by ELISA. a Number of separate experiments.

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Fig. 4. Confocal microscopic analysis of LPG and TLR-2 on NK cells. NK cells were stained with goat anti-TLR-2 Ab and a FITC-conjugated rabbit anti-goat Ab. After 3 h incubation with LPG, NK cells were stained with WIC 79.3 mAb (anti-LPG) and goat anti-mouse biotinilated Ab, followed by a streptavidin–rhodamine complex. The images were collected with a 40× oil-immersion objective lens, and were simultaneously obtained in two channels (Bio-rad confocal microscope). The merged image (yellow) indicates that TLR-2 (green) and LPG (red) co-localize in the surface of NK cells. Original magnification: 400×.

merged image (yellow), indicating that anti-TLR-2 Ab and LPG co-localize in the surface of NK cells (Fig. 4). 3.6. LPG-TLR-2 complexes are immunoprecipitated by both anti-LPG and anti-TLR-2 antibodies To analyze the binding of LPG to TLR-2 on NK cells, immunoprecipitates of stimulated and unstimulated NK

Fig. 3. Effect of Leishmania major LPG on NF-␬B activation in NK cells. NK cells were incubated in culture medium alone or in the presence of procyclic or metacyclic LPG or PMA. Following stimulation, nuclear extracts were prepared and assayed for NF-␬B translocation by Western blotting. Unstimulated NK cells were used to normalize all measurements (lane 1). The relative increase in NF-␬B translocation following PMA stimulation of NK cells is shown in lane 2. The relative increase following procyclic or metacyclic LPG stimulation is shown in lanes 3 and 4, respectively. The values in the histogram represent the average of two determinations from independent experiments. An original representative Western blot is shown in the inset.

3.4. LPG induces nuclear translocation of NF-κB in NK cells Translocation of NF-␬B to the nucleus of NK cells was analyzed after 1 h stimulation with 10 ␮g/ml metacyclic or procyclic LPG. Unstimulated NK cells showed minimal translocation, whereas the NK cells stimulated with metacyclic LPG presented a three-fold increase and the procyclic LPG presented a 2.21-fold increase. Stimulation of NK cells with 10 ng/ml PMA induced a 2.61-fold increase of nuclear translocation of NF-␬B in NK cells (Fig. 3). Degradation of cytoplasmic I␬B-␣ protein was confirmed by Western blot (data not shown). 3.5. TLR-2 and LPG co-localize in the surface of NK cells Confocal microscopical analysis of the TLR-2 expression (green) and LPG localization (red) on an NK cell shows a

Fig. 5. Immunoprecipitation of LPG stimulated and unstimulated NK cells was performed with two antibodies: the antibody against TLR-2 and the mAb against LPG (WIC 79.3) (panel A). Precipitates were subjected to Western blotting and probed with anti-LPG (lanes 1 and 2) or with anti-TLR-2 (lanes 3 and 4), respectively. The immunocomplex is observed only in NK cells stimulated with LPG (lanes 1 and 3). Uncomplexed LPG, purified from metacyclic L. major promstigotes, was blotted with WIC 79.3 (panel B, lane 2). NK cells were subjected to Western blotting and probed with anti-TLR-2 antibodies (panel C, lane 2).

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cells were made with anti-LPG mAbs and anti-TLR-2 antibodies (Fig. 5). The immunoprecipitates were then subjected to immunoblotting with anti-TLR-2 and anti-LPG, respectively. Fig. 5A shows that after immunoprecipitation with anti-TLR-2, a diffuse 175-kDa band is recognized by anti-LPG antibodies only in NK cells incubated with LPG (lane 1). After inverting the sequence of the antibodies (i.e. precipitating with anti-LPG and blotting with anti-TLR-2), again the diffuse 175-kDa band is observed only in NK cells incubated with LPG (lane 3). The specific recognition of the precipitate by both antibodies in the immunoblots reveals that LPG remains bound to TLR-2 after immunoprecipitation with either anti-TLR-2 or anti-LPG antibodies. Fig. 5B shows the immunoblotting of purified metacyclic LPG with anti-LPG mAb (WIC 79.3). Purified LPG migrates as a diffuse band spanning the region of the gel, which corresponds to a molecular weight of 20–30 kDa (lane 1). This diffuse band corresponds to the material recognized by the anti-LPG mAb in the Western blot (lane 2), which is consistent with the data reported by McConville et al. [22]. The immunoblotting of TLR-2 of NK cells (Fig. 5C) shows that the polyclonal anti-TLR-2 antibody recognizes a 83-kDa protein (lane 2), which is consistent with the data reported by the manufacturer.

4. Discussion In the present study, we investigated the ability of Leishmania LPG to trigger TLR-2 on NK cells, leading to their activation. Our results show that LPG from Leishmania is recognized by TLR-2 on NK cells, leading their activation. The immunoprecipitation experiments show that the bonds formed between TLR-2 and LPG molecules seem to be resistant to the heat and SDS treatment, leading to a high molecular weight (175 kDa) complex that migrates slowly as a diffuse band. This immune complex possibly represents an aggregate of a group of molecules that includes one TLR-2 molecule with a molecular mass of 83 kDa and perhaps 3 LPG molecules, each with a molecular mass of 30 kDa (or other unidentified molecules, and correspondingly less LPG molecules). We found that the magnitude of NK cell activation is dependent on the quantitative and qualitative changes that occur in the carbohydrate residues of the phosphosaccharide repeat units of the LPG molecule during the growth cycle of the parasite. LPG purified from the infectious metacyclic promastigotes presented a more intense NK cell activation, as compared to LPG purified from noninfectious procyclic promastigotes. The variable potency that procyclic and metacyclic LPG exerted on NK cell activation was observed in the upregulation of both mRNA and the surface expression of TLR-2 (Figs. 1 and 2), in the increased production of IFN-␥ and TNF-␣ and in the activation NF-␬B (Table 1 and Fig. 3).

Striking modifications of LPG occur during the differentiation of L. major from the noninfective procyclic stage to the highly infectious metacyclic stage. McConville et al. [26] reported an approximate doubling in the average number of phosphorylated oligosaccharide repeat units per molecule (from 14 to 30) as well as a substitution of the terminal ␤-galactose residues for ␣-arabinose and to a lesser extent ␤-glucose residues in the side chains attached to the repeat units. The relative increase in size of the metacyclic LPG is accompanied by a thickening of the surface glycocalyx by more than two-fold [8]. During this process of metacyclogenesis, there is no change in the lyso-1-O-alkylphosphatidylinositol lipid anchor or the phosphosaccharide core [27], which correspond to GPI anchors and which have previously been described as TLR-2 ligands in protozoan parasites [3]. Our data indicate that TLR-2 on NK cells recognizes carbohydrate residues on the phosphosaccharide repeat units of LPG leading to NK cell activation. This observation is strengthened by the fact that the activation effect could effectively be blocked by F(ab )2 fragments of the monoclonal mAb against L. major LPG (WIC 79.3), whose high affinity epitope is localized in the phosphosaccharide repeat units of LPG and not in the phosphosaccharide core region or lipid anchor which form part of the GPI anchor. The epitope has been mapped to the phosphorylated oligosaccharides P5b, PO4-6[Gal(␤1-3)Gal(␤1-3)Gal(␤1-3)]Gal(␤1-4)Man(␣1), unique to the LPG of promastigotes of L. major [28]. The more intense activation induced by metacyclic over procyclic LPG seems to indicate that TLR-2 possibly detects differences in the number of phosphosaccharide repeat domains and/or in the composition of glycan side chains that branch off these repeat domains. It is tempting to speculate that TLR-2 seems to ensure a more intense activation against the dangerous infective metacyclic L. major promastigotes over the nonvirulent procyclic parasites. An increased production of IFN-␥ and TNF-␣ induced by metacyclic LPG has been shown to mediate host-protection against L. major infections in the mouse model [29]. The therapeutic effect produced by TNF-␣ is due to the production of the inducible form of nitric oxide synthase (iNOS), an important leishmanicidal effector mechanism of macrophages, which is augmented by the synergistic effect of IFN-␥ [29–31]. This finding is also supported by the results of Proudfoot et al. [32] who reported that LPG obtained from stationary phase metacyclic L. major promastigotes can synergize with IFN-␥ in stimulating iNOS expression by the murine macrophage cell line J774. LPG has been shown to modulate many other host cell functions, including IL-1␤ and IL-12 production as well as CD25, LFA-1, CR3, CR4, E-selectin, CD31, ICAM-1, VCAM-1, vascular endothelium cadherin and monocyte chemoattractant protein-1 expression, [33–40]. Once inside the host cell, the metacyclic LPG repeating units also modulate several potent leishmanicidal defense mechanisms

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of the host cells such as inhibition of the phagolysosomal biogenesis [41–43]. In human NK cells, the membrane expression of TLR-1 and TLR-5 and the mRNA for TLR-2 have previously been reported [44], yet this is the first description of TLR-2 membrane expression on human NK cells. Our findings, that mRNA levels as well as membrane protein expressions of TLR-2 are upregulated in NK cells after a 24 h incubation with LPG is not surprising, since variable expressions of TLR-2 have previously been reported on monocytes exposed to LPS, GM-CSF, IL-1, IL-10 and Mycobacterium avium [45,46]. TLR-2 in NK cells seems to play an important role in activating the innate immune system and protecting the host against molecules that are able to inhibit many important leishmanicidal defense mechanisms of macrophages. Yet, even though NK cells have been shown to protect the host in the early stages of leishmaniasis [11–14], little is known about their role in later stages of the disease. In lesions of patients with diffuse cutaneous leishmaniasis, a progressive incurable form of the disease, NK cells are severely diminished and reappear after treatment and parasite reduction [47]. It will be interesting to analyze the presence of TLR-2 receptors in NK cells in patients with different clinical forms of leishmaniasis, since striking differences in cytokine and chemokine patterns as well as iNOS expression have been observed in skin biopsies of patients with localized and diffuse cutaneous leishmaniasis. Whereas in lesions of patients with localized cutaneous leishmaniasis the expression of IL-1␤, IL-6, TNF-␣, IFN-␥, macrophage chemoattractant protein (MCP-1) and iNOS was dominant, diffuse cutaneous leishmaniasis lesions were characterized by cytokines such as IL-4, IL-5, IL-10, macrophage inflammatory protein 1␣ (MIP-1␣) and a low expression of iNOS [48–51]. It is tempting to speculate that TLR-2 on NK cells possibly participates in determining the disease outcome through the induction and regulation of iNOS by IFN-␥ and TNF-␣, generating nitric oxide, which is one of the key defense mechanisms for controlling intracellular Leishmania [52]. The present results widen our knowledge of the role played by NK cells in leishmaniasis. NK cells are directly stimulated by Leishmania parasites through TLR-2, which leads to their activation enabling them to respond to Leishmania parasites in the absence of cytokines produced by other cells of the immune system. It should be interesting to identify additional Leishmania ligands for the other TLR receptors present on NK cells.

Acknowledgements We thank Dr. Ruy Pérez Becker for helpful discussions and Dr. Ruy Pérez Montfort for critical reading of the manuscript. We are indebted to Marco Elias Gudiño, Francisco Pasos and Daniel Sánchez Almaraz for technical assistance and Luc´ıa Alvarez Trejo for secretarial

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support. Additionally, we are grateful to Dr. Alejandro Ruiz Argüelles, Dr. Yvonne Rosenstein and Dr. Alejandro Padilla for their generous support.

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