TLR Ligands Set I [Toll-like Receptors Ligands Set I ]

Transcription

1 Manual TLR Ligands Set I [Toll-like Receptors Ligands Set I ] For Research Use Only APO-54N-018-KI01 Version 3 (12-Jun-08)

2 1. CONTENT The TLR Ligands Set I contains a comprehensive sterile, ready-to-use panel of agonist ligands for the study of Toll-like Receptors (mouse and human) 1 to 9 (TLRs 1 to 9). TLR1/ 2 25 µg Pam 3 CSK 4. 3HCl TLR3 1 mg Poly(I:C). potassium salt TLR4 20 µg LPS from E. coli R515 TLR5 2 µg Flagellin (purified) TLR6/ 2 2 µg MALP-2 TLR7 & µg Poly(U). potassium salt TLR9 100 µg CpG ODN 2395 QUANTITY: SHIPPING: Sufficient for at least assays. Shipped on blue ice. SHORT TERM STORAGE: -20 C. LONG TERM STORAGE: HANDLING: MANUFACTURER: -20 C. Avoid freeze/thaw cycles. Vials should be centrifuged before use to avoid loss of volume! Manufactured by Apotech Corporation. 2

3 2. INTRODUCTION Toll-like receptors (TLRs) The initial recognition of microbes during host defense is mediated by a family of receptors termed Toll-like-receptors (TLRs) [1]. Activation of the TLRs leads not only to the induction of inflammatory responses, but also to the development of antigen-specific innate and adaptive immunity [2, 3]. TLRs recognize conserved microbial components, termed pathogen-associated molecular patterns (PAMPs). To date, 12 TLRs have been cloned in mice and 10 in human and each receptor appears to be involved in the recognition of a unique set of PAMPs that are distinct in their chemical nature and structure [4]. TLRs can be divided into two categories with regard to their subcellular localisation. Cell surface TLRs includes TLR1/TLR2, TLR4 /MD2, TLR5 and TLR6 /TLR2 whereas TLR3, TLR7, TLR8 and TLR9 are found in intracellular organelles [5]. Toll-like receptors are characterized by an extracellular domain composed of leucine-rich repeats (LRRs), and an intracytoplasmic domain with a conserved region found in TLRs as well as in Interleukin-1 receptor (Toll/IL-1R or TIR domain). TLR intracellular domains specifically recruit several adaptor proteins including MyD88, TIRAP/MAL, TRIF, and TOLLIP[6]. These adaptor proteins subsequently associate with a family of Interleukin-1 Receptor-Associated Kinases (IRAK1, 2, M, and 4). Recruitments of numerous downstream signaling proteins lead to activation of a range of transcription factors such as NF-κB, AP-1, and IRFs, which are responsible for specific gene transcription [7]. TLR ligands Biochemical studies and genetic analyses using transgenic mice have revealed specific ligands for the activation of the TLR receptors. Of the 12 mouse TLRs (1 to 9 and 11 to 13) and 10 human TLRs described to date, only human TLR 10 and mouse TLR 12 and 13 are orphan receptors. TLR1, TLR2 and TLR6 (both as homo- and heterodimers) detect lipopeptide [4], while TLR3, TLR7, TLR8 and TLR9 recognize nucleic acids [5]. TLR5 senses Flagellin, a protein found in the flagella of gram-negative bacteria [8] and TLR4 recognizes a diverse collection of lipopolysaccharides (LPS) [9]. The extracellular region including the LRRs either directly or indirectly binds to their respective ligands. For instance, MD2, CD14 anf LPS-binding protein (LBP) are the co-receptors of TLR4, while flagellin, an unmethylated CpG oligonucleotides directly interact with TLR5 ad TLR9, respectively [9]. TLR11 senses the profilin-like protein from the protozoan parasite Toxoplasma gondii [10]. 3

4 References 1. Akira, S., and Takeda, K. (2004). Toll-like receptor signalling. Nat. Rev. Immunol. 4, Iwasaki, A., and Medzhitov, R. (2004). Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5, Cook, D.N., Pisetsky, D.S., and Schwartz, D.A. (2004). Toll-like receptors in the pathogenesis of human disease. Nat. Immunol. 5, Miyake, K. (2007). Innate immune sensing of pathogens and danger signals by cell surface Toll-like receptors. Sem. Immunol. 19, Krieg, A.M., and Vollmer, J. (2007). Toll-like receptors 7, 8, and 9: linking innate immunity to autoimmunity. Immunol. Rev. 220, Watters, T.M., Kenny, E.F., and O'Neill, L.A. (2007). Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. Immunol. Cell Biol. 85, Lee, M.S., and Kim, Y.J. (2007). Signaling pathways downstream of patternrecognition receptors and their cross talk. Annu. Rev. Biochem. 76, Smith, K.D., and Ozinsky, A. (2002). Toll-like receptor-5 and the innate immune response to bacterial flagellin. Curr. Top. Microbiol. Immunol. 270, Miller, S.I., Ernst, R.K., and Bader, M.W. (2005). LPS, TLR4 and infectious disease diversity. Nat. Rev. Microbiol. 3, Yarovinsky, F., Zhang, D., Andersen, J.F., Bannenberg, G.L., Serhan, C.N., Hayden, M.S., Hieny, S., Sutterwala, F.S., Flavell, R.A., Ghosh, S., et al. (2005). TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 308,

5 3. LIGANDS TLR1 ligand: Pam 3 CSK 4. 3HCl Pam 3 Cys-Ser-(Lys) 4. 3HCl [(S)-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-Npalmitoyl-(R)-Cys-(S)-Ser-(S)-Lys 4 -OH. 3HCl] Product Number: APO-54N-018/TLR1 Product Specifications: Content: 25 µg Concentration: 0.5 mg/ml Molecular weight: Purity: Formulation: Endotoxin content <0.01 EU/µg synthetic peptide as determined using the Limulus Amebocyte Lysate Assay (BioWhittaker). Liquid. Sterile, ready-to-use solution in double distilled, pyrogen-free water. Product Description: General information: Specificity: Amount per assay: Number of assays: Pam 3 CSK 4 is a synthetic tripalmitoylated lipopeptide (triacylated lipopeptide), an analog of the immunologically active N-terminal portion of bacterial lipoprotein. Pam 3 CSK 4 potently activates monocytes and macrophage. Pam 3 CSK 4 is recognized by a heterodimer formed between TLR1 and TLR2. Activation of TLR1 /TLR2 heterodimer. Stimulation of TLR1/TLR2 with Pam 3 CSK 4 can be achieved with concentrations ranging from 10 to 100 ng/ml. > 100 assays References: General Literature References: Takeuchi, O. et al. Cutting edge: role of the Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J. Immunol. 169, 10 (2002). Takeda, K. et al. Recognition of lipopeptides by Toll-like Receptors. J. Endotoxin Res. 8, 459 (2002). Sandor, F et al. Importance of extra- and intracellular domains of TLR1 and TLR2 in NF-kappa B signaling. J Cell Biol. 162, 1099 (2003). 5

6 TLR3 ligand: Poly(I:C). potassium salt Poly(I). Poly(C). potassium salt (Poly IC). potassium salt Polyinosinic-polycytidylic acid. potassium salt Product Number: APO-54N-018/TLR3 Product Specifications: Content: Concentration: Purity: Formulation: 1 mg 10 mg/ml Endotoxin content <0.01 EU/µg poly(i:c) as determined using the Limulus Amebocyte Lysate Assay (BioWhittaker). Liquid. Sterile, ready-to-use solution in double distilled, pyrogen-free water. Product Description: General information: Specificity: Amount per assay: Number of assays: Poly(I:C) is a synthetic mimetic of viral double-stranded RNA (dsrna) that is produced during replicative life cycle of a majority of viruses. The dsrna or poly(i:c) are potent inducer of type-1 Interferon (IFN) through TLR3. Poly(I:C) binds directly to TLR3. Activation of Toll-like receptor 3 (TLR3). Stimulation of TLR3 with Poly(I:C) potassium salt can be achieved with concentrations ranging from 10 to 100 µg/ml. > 50 assays References: General Literature References: Schmidt, K.N. et al. APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA. J. Immunol. 172, 138 (2004). Alexopoulou, L. et al. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413, 732 (2001). 6

7 TLR4 ligand: LPS from E. coli, Serotype R515 (Re) (liquid) Lipopolysaccharide from E. coli, Serotype R515 (Re)(liquid) Product Number: APO-54N-018/TLR4 Product Specifications: Content: 20 µg Concentration: 1 mg/ml Source: Rough (R)-form LPS isolated and purified from Escherichia coli R515 (Re mutant) by a modification of the PCP extraction method, converted to the uniform sodium salt form and dissolved in sterile distilled pyrogen-free water. No further re-extraction required. The S(smooth)-form of LPS is commonly the preferred choice for whole animal studies whereas the R(rough)- form of LPS is primarily used in cellular in vitro activation studies. Purity: Appearance: Formulation: Handling: Hazard: 99.9% (with regard to protein and DNA); Protein <0.08%. Clear solution. Liquid. Sterile, ready-to-use solution in double distilled, pyrogen-free water. Do not ingest. Wear gloves and mask when handling this compound. Avoid contact through all modes of exposure. Avoid accidental injection and inhalation and prevent this compound from entering the bloodstream. Keep sterile. Toxic. Highly pyrogenic compound. Product Description: General information: TLR4 recognizes Lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria. Recognition of LPS requires the protein CD14 in addition to TLR4. The responsiveness of TLR4/CD14 complex to LPS is enhanced by a third partner, MD-2. 7

8 Specificity: Amount per assay: Number of assays: Strong activator of Toll-like receptor (TLR) 4. LPS from most commercial sources are contaminated by other bacterial components and thus activates TLR2 as well as TLR4 signaling. LPS (serotype 515) does not activate TLR2 or other TLRs as determined with splenocytes and macrophages from TLR4 deficient mice. Stimulation of TLR4 with LPS can be achieved with concentrations ranging from 0.02 to 1 µg/ml. > 50 assays Figure: Activation of macrophages from TLR4 wild type compared to TLR4 deficient mice by LPS (Prod. No. APO-54N-018/TLR4). LPS concentration (80 ng), which induces maximal activation of TLR4 wild type mouse macrophages, was applied to TLR4 deficient mouse macrophages. 10 units of IL-6 correspond to the detection limit of the IL-6 ELISA. References: Product Specific Literature References: Galanos, C. et al. A new method for the extraction of R lipopolysaccharides. Eur. J. Biochem. 9, 245 (1969). Galanos, C and Luderitz, O. Electrodialysis of lipopolysaccharides and their conversion to uniform salt forms. Eur. J. Biochem. 54, 603 (1975). Galanos C and Luderitz, O. Isolation and purification of R-form lipopolysaccharides. Meth. Carbohydrate. Chem. 9, 11 (1993). General Literature Reference: Miller S.I. et al. LPS, TLR4 and infectious disease diversity. Nat Rev Microbiol. 1, 36 (2005). 8

9 TLR5 ligand: Flagellin (purified) FliC (purified) Product Number: APO-54N-018/TLR5 Product Specifications: Content: 2 µg Concentration: 50 µg/ml Source /Host: Isolated from Salmonella typhimurium strain Purity: Formulation: >95% (SDS-PAGE). Endotoxin content <0.1 EU/µg purified protein as determined using the Limulus Amebocyte Lysate Assay (BioWhittaker). Liquid. Sterile, ready-to-use solution in double distilled, pyrogen-free water. Product Description: General information: Specificity: Amount per assay: Number of assays: Motile bacteria produce flagella, filaments composed mainly of the structural subunit flagellin. Flagellins are highly conserved molecules among both gram negative and gram positive bacteria, especially in 170 N-terminal and 100 C-terminal amino acids. In mammalian cells, Toll-like receptor 5 (TLR5) senses various flagellins resulting in activation of innate and adaptive immunity. The flagellin FliC from Salmonella typhimurium (serotype H 'I'), a protein of 494 amino acids, upregulates production of chemokines and antimicrobial compounds in human epithelial cells and monocytes and induces maturation of dendritic cells. Activation of Toll-like receptor 5 (TLR5). Stimulation of TLR5 with Flagellin can be achieved with concentrations ranging from 10 to 100 ng/ml. > 50 assays 9

10 Method: Activation of TLR5 in human epithelial cell assays based on NF-kB luciferase fusion, on expression of IL-8 (CXCL-8), CCL20. Salmonella-derived flagellin (Prod. No. APO-54N-018/TLR5) upregulates CCL20 and IL-8 transcription in epithelial intestinal cells. Epithelial intestinal Caco-2 cells were treated 3.30h with flagellin at the indicated concentrations. CCL20 and IL-8 gene transcription was quantified by real-time RT-PCR. References: Product Specific Literature Reference: Sierro F. et al. Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells: PNAS 98, (2001). General Literature References: Hayashi et al. The innate immune response to bacterial flagellin is mediated by Toll- Like receptor 5. Nature 410, 1099 (2001). Gewirtz A.T. et al. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J.Immunol. 167, 1882 (2001). 10

11 TLR6 ligand: MALP-2 Macrophage stimulatory Lipopeptide 2 Macrophage-activating Lipopeptide-2 S-(2,3- bisacyloxypropyl)-cysteine-gnndesnisfkek Product Number: APO-54N-018/TLR6 Product Specifications: Content: 2 µg Concentration: 0.1 mg/ml Molecular weight: 2136 Source /Host: Purity: Formulation: Use : Synthetic >98% (SDS-PAGE). Endotoxin content <0.01 EU/µg purified protein as determined using the Limulus Amebocyte Lysate Assay (BioWhittaker). Liquid. Sterile, ready-to-use solution in PBS containing 25 mm n-octyl-β-d-glucopyranoside and 1% human serum albumin. Since MALP-2 is active at very low concentrations, i.e. at high dilutions, these low amounts of material tend to become adsorbed to pipette tips and plastic or glass containers. To avoid this, prepare several dilution steps of the MALP-2 stock solution with medium containing 5% serum or other protective protein. Because of the ester-bound fatty acids, MALP-2 is sensitive to alkali. The medium should be preconditioned in the CO2 incubator in order to ensure that the ph does not become alkaline. Add cells in preconditioned medium as soon as possible and incubate. Handling: Toxic compound. Avoid accidental injection and inhalation and prevent this compound from entering the bloodstream. 11

12 Product Description: General information: Specificity: Amount per assay: Number of assays: MALP-2 is a well-defined diacylated lipopeptide isolated from Mycoplasma fermentans. MALP-2 is recognized by TLR6 only when heterodimerized with TLR2. CD36, a member of the scavenger receptor type B family, acts as a facilitator or co-receptor for MALP-2 recognition through the TLR2/6 complex. Activation of Toll-like receptor 6 (TLR6) complexed with TLR2. Stimulation of TLR6 /TLR2 with MALP-2 can be achieved with concentrations ranging from 10 to 100 ng/ml. > 100 assays References: Product Specific Literature References: Mühlradt P.F. et al.;. Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplama fermentans acting at picomolar concentration: J. Exp. Med. 185, 1951 (1997). Galanos C. et al. MALP-2, a Mycoplasma lipopeptide with classical endotoxic properties: end of an era of LPS monopoly? J. Endotox. Res. 6, 471 (2000). Takeuchi, O. et al. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int. Immunol. 13, 933 (2001). Satta N. et al. The role of TLR2 in the inflammatory activation of mouse fibroblasts by human antiphospholipid Antibodies. Blood 109, 1507 (2007). General Literature References: Into, T. et al. Synergic effectgs of mycoplasmal lipopeptides and extracellular ATP on activation of macrophages. Infect. Immun. 70, 3586 (2002). Davis K.L. and Wise K.S. Site-specific proteolysis of the MALP-404 lipoprotein determines the release of a soluble selective lipoprotein-associated motif-containing fragment and alteration of the surface phenotype of Mycoplasma fermentansinfect. Immun. 70, 1129 (2002). 12

13 TLR7 & 8 ligand: Poly(U) potassium. salt Polyuridylic acid. potassium salt Product Number: APO-54N-018/TLR7-8 Product Specifications: Content: 100 µg Concentration: Source: 1 mg/ml Synthetic analog of single-stranded RNA (ssrna). Purity: Endotoxin content <0.1 EU/µg purified synthetic poly(u)(polyuridylic acid) as determined using the Limulus Amebocyte Lysate Assay (BioWhittaker). Formulation: Liquid. Sterile, ready-to-use solution in double distilled, pyrogen-free water. Product Description: General information: Specificity: Activation of TLR7 and 8. Poly(U) is a simple motif single stranded RNA (ssrna). Uridine (U)-rich ssrnas are recognized by TLR7 (murine) and TLR8 (human). This motif is likely present in viral as well as non-viral RNAs (self RNAs). TLR7 and 8, which are localized in the endosomes, probably detect only RNAs with abnormal localization. In most cases, RNAs present in endosomal compartments are of viral origin (self RNAs are degraded by extracellular ribonucleases). Amount per assay: Number of assays: Stimulation of TLR7 or 8 with poly (U) can be achieved with concentrations ranging from 0.1 to 10 µg/ml. > 100 assays Reference: General Literature Reference: Diebold S.S. et al. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA: Science 303, 1529 (2004). 13

14 TLR9 ligand: CpG ODN 2395 Product Number: APO-54N-018/TLR9-C100 Product Specifications: Content: 100 µg Concentration: Purity: Formulation: 0.7 mg/ml Endotoxin content <0.1 EU/µg CpG ODN 2395 as determined using the Limulus Amebocyte Lysate Assay (BioWhittaker). Liquid. Sterile, ready-to-use solution in double distilled, pyrogen-free water. Product Description: General information: Synthetic oligodeoxynucleotides (ODN) containing unmethylated deoxycytosine-deoxyguanosine (CpG) motifs are very potent inducers of the innate immune system, mimicking the effects of bacterial DNA. CpG ODNs are recognized by Toll-like receptor 9 (TLR9). CpG ODN 2395 belongs to the recently described C- Class ODN that most efficiently combines the properties of A- and B-Class ODN in that they induce strong B-cell activation comparable to B-Class ODN together with IFN-alpha secretion comparable to A-Class ODN. CpG ODN 2395 consists of a 5' stimulatory hexameric CpG motif linked by a T spacer to 3' palindromic sequences that are preferably GC-rich. Sequence: Specificity: TCG TCG TTT TCG GCG CGC GCC G, fully phosphorothioate backbone. Activation of murine and human TLR9. Amount per assay: Number of assays: Stimulation of TLR9 with CpG ODN 2395 can be achieved with concentrations ranging from 0.1 to 10 µg/ml. > 50 assays 14

15 Figure: CpG ODN 2395 (Prod. No. APO-54N-018/TLR9)-induced activation of Toll-like receptor 9. Method: 10 5 murine splenocytes were incubated with the indicated concentrations of CpG ODN 2395 or with the non CpG ODN After 18 hours, supernatants were harvested and tested on the TNF-sensitive cell line WEHI 164. TNF-a production was measured, using TNF-α, Soluble (human) (rec.) (Prod. No. APO-50N- 001-C050) as standard. References: General Literature Reference: Vollmer J. et al. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur. J. Immul. 34, 251 (2004). 15

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