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Physicochemical and Biological Analysis of Synthetic Bacterial Lipopeptides

2007; Elsevier BV; Volume: 282; Issue: 15 Linguagem: Inglês

10.1074/jbc.m700287200

1083-351X

Andra B. Schromm, Jörg Howe, Artur J. Ulmer, Karl‐Heinz Wiesmüller, Tobias Seyberth, Günther Jung, Manfred Rößle, Michel H. J. Koch, Thomas Gutsmann, Klaus Brandenburg,

Natural product bioactivities and synthesis

The importance of the biological function and activity of lipoproteins from the outer or cytoplasmic membranes of Gram-positive and Gram-negative bacteria is being increasingly recognized. It is well established that they are like the endotoxins (lipopolysaccharide (LPS)), which are the main amphiphilic components of the outer membrane of Gram-negative bacteria, potent stimulants of the human innate immune system, and elicit a variety of proinflammatory immune responses. Investigations of synthetic lipopeptides corresponding to N-terminal partial structures of bacterial lipoproteins defined the chemical prerequisites for their biological activity and in particular the number and length of acyl chains and sequence of the peptide part. Here we present experimental data on the biophysical mechanisms underlying lipopeptide bioactivity. Investigation of selected synthetic diacylated and triacylated lipopeptides revealed that the geometry of these molecules (i.e. the molecular conformations and supramolecular aggregate structures) and the preference for membrane intercalation provide an explanation for the biological activities of the different lipopeptides. This refers in particular to the agonistic or antagonistic activity (i.e. their ability to induce cytokines in mononuclear cells or to block this activity, respectively). Biological activity of lipopeptides was hardly affected by the LPS-neutralizing antibiotic polymyxin B, and the biophysical interaction characteristics were found to be in sharp contrast to that of LPS with polymyxin B. The analytical data show that our concept of "endotoxic conformation," originally developed for LPS, can be applied also to the investigated lipopeptide and suggest that the molecular mechanisms of cell activation by amphiphilic molecules are governed by a general principle. The importance of the biological function and activity of lipoproteins from the outer or cytoplasmic membranes of Gram-positive and Gram-negative bacteria is being increasingly recognized. It is well established that they are like the endotoxins (lipopolysaccharide (LPS)), which are the main amphiphilic components of the outer membrane of Gram-negative bacteria, potent stimulants of the human innate immune system, and elicit a variety of proinflammatory immune responses. Investigations of synthetic lipopeptides corresponding to N-terminal partial structures of bacterial lipoproteins defined the chemical prerequisites for their biological activity and in particular the number and length of acyl chains and sequence of the peptide part. Here we present experimental data on the biophysical mechanisms underlying lipopeptide bioactivity. Investigation of selected synthetic diacylated and triacylated lipopeptides revealed that the geometry of these molecules (i.e. the molecular conformations and supramolecular aggregate structures) and the preference for membrane intercalation provide an explanation for the biological activities of the different lipopeptides. This refers in particular to the agonistic or antagonistic activity (i.e. their ability to induce cytokines in mononuclear cells or to block this activity, respectively). Biological activity of lipopeptides was hardly affected by the LPS-neutralizing antibiotic polymyxin B, and the biophysical interaction characteristics were found to be in sharp contrast to that of LPS with polymyxin B. The analytical data show that our concept of "endotoxic conformation," originally developed for LPS, can be applied also to the investigated lipopeptide and suggest that the molecular mechanisms of cell activation by amphiphilic molecules are governed by a general principle. Beside lipopolysaccharides (LPS, 2The abbreviations used are: LPS, lipopolysaccharide; FRET, fluorescence resonance energy transfer; IL, interleukin; LBP, lipopolysaccharide-binding protein; lipolan, lipolanthionine peptide (2R,6R)Pam2LanHda-Ser-(Lys)4-NH2; LP, lipopeptide; Pam2CSK4, dipalmitoyl-S-glyceryl-l-Cys-Ser-(Lys)4; Pam3CSK4, tripalmitoyl-S-glyceryl-l-Cys-Ser-(Lys)4; PMB, polymyxin B; TLR, Toll-like receptor; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE; Rh-PE, N-(rhodamine B sulfonyl)-PE; PL, phospholipid; hu, human. 2The abbreviations used are: LPS, lipopolysaccharide; FRET, fluorescence resonance energy transfer; IL, interleukin; LBP, lipopolysaccharide-binding protein; lipolan, lipolanthionine peptide (2R,6R)Pam2LanHda-Ser-(Lys)4-NH2; LP, lipopeptide; Pam2CSK4, dipalmitoyl-S-glyceryl-l-Cys-Ser-(Lys)4; Pam3CSK4, tripalmitoyl-S-glyceryl-l-Cys-Ser-(Lys)4; PMB, polymyxin B; TLR, Toll-like receptor; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE; Rh-PE, N-(rhodamine B sulfonyl)-PE; PL, phospholipid; hu, human. endotoxin), which are the major amphiphilic components of the outer membrane of Gram-negative bacteria, other amphiphilic molecules such as lipoproteins and lipopeptides (LP) are found in the cell wall of a large number of microorganisms, including Gram-negative as well as Gram-positive bacteria, mycobacteria, mycoplasms, and spirochetes (1Gioffre A. Caimi K. Zumarraga M.J. Meikle V. Morsella C. Bigi F. Alito A. Santangelo M.P. Paolicchi F. Romano M.I. Cataldi A. J. Vet. Med. Ser. B. 2006; 53: 34-41Crossref PubMed Scopus (6) Google Scholar, 2Wardenburg J.B. Williams W.A. Missiakas D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 13831-13836Crossref PubMed Scopus (178) Google Scholar, 3Hashimoto M. Tawaratsumida K. Kariya H. Kiyohara A. Suda Y. Krikae F. Kirikae T. Gotz F. J. Immunol. 2006; 177: 3162-3169Crossref PubMed Scopus (198) Google Scholar, 4Kataoka H. Yasuda M. Iyori M. Kiura K. Narita M. Nakata T. Shibata K. Cell. Microbiol. 2006; 8: 1199-1209Crossref PubMed Scopus (24) Google Scholar, 5Yang X.F. Alani S.M. Norgard M.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11001-11006Crossref PubMed Scopus (170) Google Scholar). Like LPS these molecules can be potent activators of lymphocytes and macrophages and give danger signals to the infected host (6Melchers F. Braun V. Galanos C. J. Exp. Med. 1975; 142: 473-482Crossref PubMed Scopus (186) Google Scholar, 7Sellati T.J. Waldrop S.L. Salazar J.C. Bergstresser P.R. Picker L.J. Radolf J.D. J. Immunol. 2001; 166: 4131-4140Crossref PubMed Scopus (41) Google Scholar). A variety of biological activities have been described for native LP as well as for synthetic LP based on the highly conserved lipopeptide moiety of Braun's lipoprotein derived from Escherichia coli (8Braun V. Wolff H. Eur. J. Biochem. 1970; 14: 387-391Crossref PubMed Scopus (90) Google Scholar). The innate immune system plays an essential role in the host defense against bacterial infection. Recognition of bacterial virulence factors is mandatory for the initiation of an inflammatory and anti-bacterial immune response. Bacterial compounds such as LPS and lipoproteins are recognized by type I transmembrane proteins of the Toll-like receptor (TLR) family (9Underhill D.M. Ozinsky A. Curr. Opin. Immunol. 2002; 14: 103-110Crossref PubMed Scopus (591) Google Scholar). Of the more than 10 known TLRs, several have been found to participate in the recognition of microbial infections. TLR4 initiates cell activation by LPS in concert with the extracellular protein MD-2 (10Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. RicciardiCastagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6438) Google Scholar, 11Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1748) Google Scholar, 12Schromm A.B. Lien E. Henneke P. Chow J.C. Yoshimura A. Heine H. Latz E. Monks B.G. Schwartz D.A. Miyake K. Golenbock D.T. J. Exp. Med. 2001; 194: 79-88Crossref PubMed Scopus (240) Google Scholar). TLR4/MD-2 has been found to associate in complexes that include a variety of accessory proteins such as CD14, CD11/CD18, CD55, hsp70, hsp90, GDF5, and CXCR4 (13Delude R.L. Savedra Jr., R. Zhao H. Thieringer R. Yamamoto S. Fenton M.J. Golenbock D.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9288-9292Crossref PubMed Scopus (172) Google Scholar, 14Blondin C. Le Dur A. Cholley B. Caroff M. Haeffner-Cavaillon N. Eur. J. Immunol. 1997; 27: 3303-3309Crossref PubMed Scopus (30) Google Scholar, 15Correia J.D. Soldau K. Christen U. Tobias P.S. Ulevitch R.J. J. Biol. Chem. 2001; 276: 21129-21135Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar, 16Kielian T.L. Blecha F. Immunopharmacology. 1995; 29: 187-205Crossref PubMed Scopus (142) Google Scholar, 17El-Samalouti V.T. Schletter J. Chyla I. Lentschat A. Mamat U. Brade L. Flad H.-D. Ulmer A.J. Hamann L. FEMS Immunol. Med. Microbiol. 1999; 23: 259-269Crossref PubMed Google Scholar, 18Triantafilou K. Triantafilou M. Dedrick R.L. Nat. Immun. 2001; 2: 338-345Crossref Scopus (355) Google Scholar, 19Triantafilou M. Brandenburg K. Gutsmann T. Seydel U. Triantafilou K. Crit. Rev. Immunol. 2002; 22: 251-268Crossref PubMed Google Scholar, 20Triantafilou M. Miyake K. Golenbock D.T. Triantafilou K. J. Cell Sci. 1999; 115: 2603-2611Crossref Google Scholar), MOE-SIN (21Iontcheva I. Amar S. Zawawi K.H. Kantarci A. Van Dyke T.E. Infect. Immun. 2004; 72: 2312-2320Crossref PubMed Scopus (68) Google Scholar), the potassium channel MaxiK (22Blunck R. Scheel O. Mueller M. Brandenburg K. Seitzer U. Seydel U. J. Immunol. 2001; 166: 1009-1015Crossref PubMed Scopus (125) Google Scholar), and a membrane-associated form of LBP (23Gutsmann T. Mueller M. Carroll S.F. MacKenzie R.C. Wiese A. Seydel U. Infect. Immun. 2001; 69: 6942-6950Crossref PubMed Scopus (167) Google Scholar, 24Mueller M. Scheel O. Lindner B. Gutsmann T. Seydel U. J. Endotoxin Res. 2002; 9: 181-186Crossref Google Scholar). For TLR2, a variety of different ligands has been described, including lipoteichoic acid, lipoarabinomannan, bacterial lipoproteins/lipopeptides, and also some LPS variants from Gram-negative bacteria, yeast, spirochetes, and fungi (25Heine H. Lien E. Int. Arch. Allergy Immunol. 2003; 130: 180-192Crossref PubMed Scopus (126) Google Scholar). TLR2 has also been shown to associate with coreceptors (26Ozinsky A. Underhill D.M. Fontenot J.D. Hajjar A.M. Smith K.D. Wilson C.B. Schroeder L. Aderem A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13766-13771Crossref PubMed Scopus (1681) Google Scholar). Together with Dectin-1, it recognizes infections by yeasts (27Gantner B.N. Simmons R.M. Canavera S.J. Akira S. Underhill D.M. J. Exp. Med. 2003; 197: 1107-1117Crossref PubMed Scopus (1324) Google Scholar, 28Viriyakosol S. Fierer J. Brown G.D. Kirkland T.N. Infect. Immun. 2005; 73: 1553-1560Crossref PubMed Scopus (153) Google Scholar) and mycobacteria (29Yadav M. Schorey J.S. Blood. 2006; 108: 3168-3175Crossref PubMed Scopus (324) Google Scholar). Via heteromeric receptor complexes with TLR1 and -6, TLR2 also recognizes a variety of bacterial lipopeptides (30Buwitt-Beckmann U. Heine H. Wiesmueller K.H. Jung G. Brock R. Ulmer A.J. FEBS J. 2005; 272: 6354-6364Crossref PubMed Scopus (95) Google Scholar, 31Buwitt-Beckmann U. Heine H. Wiesmuller K.H. Jung G. Brock R. Akira S. Ulmer A.J. J. Biol. Chem. 2006; 281: 9049-9057Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 32Liu H. Komai-Koma M. Xu D. Liew F.Y. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 7048-7053Crossref PubMed Scopus (409) Google Scholar). Recently, it was suggested that CD36 may have a role in the recognition of the diacylated bacterial lipopeptide MALP-2 (33Hoebe K. Georgel P. Rutschmann S. Du X. Mudd S. Crozat K. Sovath S. Shamel L. Hartung T. Zaehringer U. Beutler B. Nature. 2005; 433: 523-527Crossref PubMed Scopus (706) Google Scholar). As far as the structural prerequisites for bioactivity of lipopeptides are concerned, it has been shown that tripalmitoyl-S-glyceryl-l-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4) was highly active, whereas the natural dipeptide part of the outer membrane lipoprotein, tripalmitoyl-S-glyceryl-l-Cys-Ser (Pam3CS), only has a low activity (34Bessler W.G. Cox M. Lex A. Suhr B. Wiesmuller K.H. Jung G. J. Immunol. 1985; 135: 1900-1905PubMed Google Scholar, 35Prass W. Ringsdorf H. Bessler W. Wiesmuller K.H. Jung G. Biochim. Biophys. Acta. 1987; 900: 116-128Crossref PubMed Scopus (71) Google Scholar). It is known for many amphiphilic molecules like phospholipids and glycolipids that physical parameters are strong determinants of biological activity. Above a certain threshold concentration, i.e. the critical micellar concentration, these molecules form aggregates in aqueous dispersions, which may adopt different phase states as follows: an ordered gel phase, where the acyl chains are in the all-trans (zig-zag) conformation, and a liquid crystalline phase, where the chains are disordered as a result of the introduction of gauche conformers. Between these phases a reversible first order transition takes place at a lipid-specific temperature. Furthermore, the type of aggregate structure, uni- and multilamellar, cubic direct or inverted, hexagonal HI or inverted HII, may play a decisive role in biological systems (36Israelachvili J.N. Intermolecular and Surface Forces.2nd Ed. Academic Press, London1991: 366-394Google Scholar). It has been shown for LPS and its lipid moiety, lipid A, the endotoxic principle of LPS (37Rietschel E.T. Brade H. Sci. Am. 1992; 267: 54-61Crossref PubMed Scopus (456) Google Scholar), that the aggregation type is essential for the expression of bioactivity, and that the state of order of the acyl chains may be a modulator of bioactivity (38Brandenburg K. Mayer H. Koch M.H.J. Weckesser J. Rietschel E.Th. Seydel U. Eur. J. Biochem. 1993; 218: 555-563Crossref PubMed Scopus (155) Google Scholar, 39Brandenburg K. Wiese A. Curr. Top. Med. Chem. 2004; 4: 1127-1146Crossref PubMed Scopus (111) Google Scholar). For lipopeptides, only for the immunoadjuvants Pam3-l-Cys and Pam3-l-Cys-Ser, experimental data are available, indicating that these molecules form vesicular or tubular aggregates of different sizes in aqueous dispersion depending on the configuration of the glycerol moiety and the head group (40Reichel F. Roelofsen A.M. Geurts H.P. Der Gaast S.J. Feiters M.C. Boons G.J. J. Org. Chem. 2000; 65: 3357-3366Crossref PubMed Scopus (15) Google Scholar). Furthermore, two Pam3Cys-peptides LP1 and LP2, characterized by fluorescence correlation spectroscopy, form large and highly heterogeneous aggregates where the smaller aggregates were shown to have higher bioactivity. 3A. J. Ulmer, K.-H. Wiesmüller, T. Seyberth, and G. Jung, unpublished observations. 3A. J. Ulmer, K.-H. Wiesmüller, T. Seyberth, and G. Jung, unpublished observations. To clarify the correlation between physicochemical characteristics and biological activities, we have investigated three selected lipopeptides with different cytokine-inducing activity, with strong agonistic as well as antagonistic activity in human mononuclear cells. We have found that as in the case of endotoxins, the types of aggregate structures are important determinants of cytokine-inducing activity. Furthermore, we show that our concept of "endotoxic conformation," which was shown to be applicable so far not only to LPS but also to particular synthetic phospholipids (41Seydel U. Hawkins L. Schromm A.B. Heine H. Scheel O. Koch M.H. Brandenburg K. Eur. J. Immunol. 2003; 33: 1586-1592Crossref PubMed Scopus (78) Google Scholar, 42Brandenburg K. Hawkins L. Garidel P. Andrä J. Müller M. Heine H. Koch M.H.J. Seydel U. Biochemistry. 2004; 43: 4039-4046Crossref PubMed Scopus (34) Google Scholar), can also be extended to the lipopeptides. In addition, we present data showing that a biologically inactive lipopeptide, which antagonizes cell activation by biologically active lipopeptides, is also able to antagonize cell activation by LPS, supporting the broad validity of the conformation concept for agonistic as well as antagonistic activity. Synthesis—Pam2CSK4 and Pam3CSK4 were synthesized and analyzed by EMC microcollections GmbH according to published procedures (43Metzger J. Wiesmuller K.H. Schaude R. Bessler W.G. Jung G. Int. J. Pept. Protein Res. 1991; 37: 46-57Crossref PubMed Scopus (74) Google Scholar, 44Wiesmueller K.H. Bessler W.G. Jung G. Int. J. Pept. Protein Res. 1992; 40: 255-260Crossref PubMed Scopus (79) Google Scholar, 45Metzger J.W. Beck-Sickinger A.G. Loleit M. Eckert M. Bessler W.G. Jung G. J. Pept. Sci. 1995; 1: 184-190Crossref PubMed Scopus (41) Google Scholar). The synthesis and analysis of the lipolanthionine peptide are described elsewhere in detail (46Seyberth T. Voss S. Brock R. Wiesmueller K.H. Jung G. J. Med. Chem. 2006; 49: 1754-1765Crossref PubMed Scopus (26) Google Scholar). The structural formula of the synthetic compounds dipalmitoyl-S-glyceryl-l-Cys-Ser-(Lys)4 (Pam2CSK4), tripalmitoyl-S-glyceryl-l-Cys-Ser-(Lys)4 (Pam3CSK4), and of the lipolanthionine peptide (2R,6R)-Pam2LanHda-Ser-(Lys)4-NH2 (lipolan) are shown in Fig. 1. The stereochemistry and composition of the lipid tails of lipolan have been optimized for the inhibition of TLR2-dependent IL-8 induction by Pam3CSK4 in myelomonocytic THP-1 cells (46Seyberth T. Voss S. Brock R. Wiesmueller K.H. Jung G. J. Med. Chem. 2006; 49: 1754-1765Crossref PubMed Scopus (26) Google Scholar). Reagents—Deep rough mutant LPS (Re LPS) was extracted from Salmonella enterica sv. Minnesota strain R595 according to the phenol/chloroform/petrol ether procedure (47Galanos C. Lüderitz O. Westphal O. Eur. J. Biochem. 1969; 9: 245-249Crossref PubMed Scopus (1366) Google Scholar). The LPS preparation was lyophilized and used in the natural salt form. The chemical purity of the LPS preparation was confirmed by mass spectrometry. LPS was suspended in PBS (Biochrom, Berlin, Germany) by thorough vortexing. The suspensions were temperature-cycled at least twice between 4 and 56 °C, each cycle being followed by intense vortexing for a few minutes and then stored at 4 °C for at least 12 h prior to measurement. Suspensions were aliquoted and stored at –20 °C. Phosphatidylcholine, sphingomyelin, and phosphatidylserine from bovine brain and phosphatidylethanolamine (PE) from E. coli were purchased from Avanti Polar Lipids (Alabaster, AL). All phospholipids were used without further purification. The fluorescent dyes N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE (NBD-PE) and N-(rhodamine B sulfonyl)-PE (Rh-PE) were purchased from Molecular Probes (Eugene, OR). Recombinant human LBP (456 amino acid holoprotein rLBP50) in 10 mm HEPES, pH 7.5, was a kind gift of XOMA LLC (Berkeley, CA). Lipid Sample Preparation—All lipid samples were prepared as aqueous suspensions in 20 mm HEPES, pH 7. For this, the lipids were suspended directly in buffer, thoroughly vortexed, temperature-cycled three times between 4 and 56 °C to enable the formation of stable aggregates and then stored for at least 12 h before measurement. To guarantee physiological conditions, the water content of the samples was usually around 95%. For preparations of liposomes from a mixture corresponding to the phospholipid composition of the macrophage membrane (phosphatidylcholine, phosphatidylserine, PE, and sphingomyelin in a molar ratio of 1:0.4:0.7:0.5), the lipids were solubilized in chloroform; the solvent was evaporated under a stream of nitrogen, and the lipids were resuspended in the appropriate volume of PBS, pH 7.0, and temperature-cycled as described above. Electron microscopy (kindly performed by H. Kühl, Division of Pathology, Forschungszentrum Borstel) revealed large multilamellar liposomes. Activation of Human Mononuclear Cells and Macrophages—Mononuclear cells were isolated from human peripheral blood of healthy donors by the Ficoll-Histopaque gradient method and cultivated at 37 °C with 6% CO2 in Teflon bags in RPMI 1640 medium (endotoxin ≤0.01 EU/ml; Biochrom, Berlin, Germany) containing 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, and 4% heat-inactivated human serum type AB from healthy donors. Cells were cultured in the presence of 2 ng/ml macrophage colony-stimulating factor for 7 days to differentiate monocytes to macrophages. To determine cytokine induction after cell stimulation, mononuclear cells were seeded in 200-μl aliquots of a suspension of 5 × 106 cells/ml, and macrophages were seeded at 1 × 106 cells/ml in 96-well tissue culture dishes (Nunc, Wiesbaden, Germany) in RPMI 1640 medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, with or without 4% human serum, and stimuli were added as indicated in the respective experiments. Cell-free supernatants were collected 4 h after stimulation and stored at –20 °C until determination of cytokine content. Data shown are mean ± S.D. of triplicate samples of one experiment and representative of at least three independent experiments. Antagonistic Action of the Inactive Lipopeptide—Inactive lipopeptides, i.e. compounds that did not induce any cytokines in human mononuclear cells, were investigated with respect to their ability to block the LPS-induced TNFα production in human macrophages. For this, Re LPS from S. minnesota R595 was prepared in three concentrations of 0.5, 1, and 5 ng/ml, and the agonistically inactive lipopeptide was added at a concentration of 1 μg/ml, and the TNFα production of the macrophages was determined. Transient Transfection and Stimulation of HEK293 Cells—HEK293 cells were plated at a density of 1.5 × 105 cells/ml in 96-well plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 0.5 unit/ml penicillin, and 0.5 μg/ml streptomycin. The following day, cells were transiently transfected using Polyfect (Qiagen, Hilden, Germany) according to the manufacturer's protocol and plasmids containing huTLR4, huMD2, and/or huCD14 as described elsewhere (30Buwitt-Beckmann U. Heine H. Wiesmueller K.H. Jung G. Brock R. Ulmer A.J. FEBS J. 2005; 272: 6354-6364Crossref PubMed Scopus (95) Google Scholar). After 6 h of transfection cells were washed and stimulated with LPS at the indicated concentrations in the absence or presence of 10 μm lipolan for a further 24 h. Cell-free supernatants were collected and stored at –20 °C until determination of cytokine content. Data shown are the mean ± S.D. of triplicate samples of one experiment and representative of at least three independent experiments. Cytokine Determination—Human TNFα was determined in pooled cell-free supernatants of stimulated cells by sandwich enzyme-linked immunosorbent assay using monoclonal mouse antibody against human TNFα and peroxidase-conjugated rabbit anti-human TNFα antibody, respectively (Intex, Muttant, Switzerland), as stated in detail elsewhere (48Mueller M. Brandenburg K. Dedrick R. Schromm A.B. Seydel U. J. Immunol. 2005; 174: 1091-1096Crossref PubMed Scopus (62) Google Scholar). Human IL-8 was determined by sandwich enzyme-linked immunosorbent assay using IL-8 cytoset from BIOSOURCE exactly according to the manufacturer's protocol. Data shown are mean ± S.D. of triplicate samples of one representative experiment. Fluorescence Resonance Energy Transfer Spectroscopy—The fluorescence resonance energy transfer (FRET) technique was used as a probe dilution assay (49Schromm A.B. Brandenburg K. Rietschel E.Th. Flad H.-D. Carroll S.F. Seydel U. FEBS Lett. 1996; 399: 267-271Crossref PubMed Scopus (111) Google Scholar) to obtain information on the intercalation of synthetic lipopeptides into liposomes resembling the lipid composition of the cytoplasmic membrane of macrophages (PLMΦ). For the FRET experiments, liposomes were double-labeled with NBD-PE and Rh-PE in chloroform [PL]:[NBD-PE]:[Rh-PE] at 100:1:1 molar ratios. The solvent was evaporated under a stream of nitrogen, and the lipids were resuspended in PBS, mixed thoroughly, and sonicated with a Branson sonicator for 1 min (1 ml of solution). Subsequently, the preparation was temperature-cycled at least twice between 4 and 56 °C, and each cycle was followed by intense vortexing for a few minutes and then stored at 4 °C for at least 12 h prior to measurement. A preparation of 900 μl of the double-labeled liposomes (10–5 m) at 37 °C was excited at 470 nm (excitation wavelength of NBD-PE), and the intensities of the emission light of the donor NBD-PE (531 nm) and acceptor Rh-PE (593 nm) were measured simultaneously on the fluorescence spectrometer SPEX F1T11 (SPEX Instruments, Edison, NY). Compounds Pam2CSK4, Pam3CSK4, and lipolan (concentration 1 μm) were added to liposomes after 50 s. Because FRET spectroscopy is used here as a probe dilution assay, intercalation of unlabeled molecules causes an increase of the distance between donor and acceptor and thus leads to a reduced energy transfer. This again causes an increase of the donor and decrease of the acceptor intensities. For a qualitative analysis of experiments, the ratio of the intensities of the donor dye and the acceptor dye are plotted against time (denoted in the following as the FRET signal). The data shown are representative for three independent experiments. Fourier Transform Infrared Spectroscopy—The infrared spectroscopic measurements were performed on an IFS-55 spectrometer (Bruker, Karlsruhe, Germany). For phase transition measurements, the lipid samples were placed between CaF2 windows with a 12.5-μm Teflon spacer. Temperature scans were performed automatically between –10 and 70 °C with a heating rate of 0.6 °C/min. Every 3 °C, 50 interferograms were accumulated, apodized, Fourier-transformed, and converted to absorbance spectra. For the identification of particular functional groups, infrared spectra of the lipopeptides at 95% water concentration were analyzed. The vibrational bands typical for the hydrophobic region (symmetrical and antisymmetrical) νs and νas stretching vibration of –CH2–groups around 2920 and 2850 cm–1, respectively, in the IR spectra of lipopeptides are sensitive markers of acyl chain order. X-ray Diffraction—X-ray diffraction measurements were performed at the European Molecular Biology Laboratory (EMBL) outstation at the Hamburg synchrotron radiation facility HASYLAB using the SAXS camera X33 (50Koch M.H.J. Makromol. Chem. Macromol. Symp. 1988; 15: 79-90Crossref Scopus (25) Google Scholar). Diffraction patterns in the range of the scattering vector 0.1 < s < 1.0 nm–1 (s = 2 sin θ/λ, 2θ scattering angle and λ the wavelength = 0.15 nm) were recorded at 40 °C with exposure times of 1 min using an image plate detector with on-line readout (MAR345, MarResearch, Norderstedt, Germany). The s axis was calibrated with Ag-Behenate, which has a periodicity of 58.4 nm. The diffraction patterns were evaluated as described previously (51Brandenburg K. Richter W. Koch M.H.J. Meyer H.W. Seydel U. Chem. Phys. Lipids. 1998; 91: 53-69Crossref PubMed Scopus (76) Google Scholar) assigning the spacing ratios of the main scattering maxima to defined three-dimensional structures. The lamellar and cubic structures are most relevant here. They are characterized by the following features. 1) Lamellar, the reflections are grouped in equidistant ratios, i.e. 1, 1/2, 1/3, 1/4, etc. of the lamellar repeat distance dl. 2) Cubic, the different space groups of these nonlamellar three-dimensional structures differ in the ratio of their spacings. The relation between reciprocal spacing shkl = 1/dhkl and lattice constant a is as follows: shkl = ((h2 + k2 + l2)/a)1/2, where hkl = Miller indices of the corresponding set of plane. In the case of very weak diffraction maxima, resolution enhancement techniques were applied, in particular Fourier self-deconvolution as described by Kauppinen et al. (52Kauppinen J.K. Moffat D.J. Mantsch H.H. Cameron D.G. Appl. Spectrosc. 1981; 35: 271-276Crossref Google Scholar). Induction of TNFα in Mononuclear Cells by Synthetic Lipopeptides—The ability of Pam2CSK4 and Pam3CSK4 to induce the production of TNFα in human mononuclear cells was tested in comparison with LPS deep rough mutant Re (from strain R595 of Salmonella minnesota) (Fig. 2). Clearly, Re LPS is active down to 100 pg/ml, whereas the lipopeptides are active at least down to 10 ng/ml. The lipolanthionine peptide lipolan was completely inactive with respect to cytokine induction up to a concentration of 10 μg/ml (data not shown). Synthetic Lipolanthionine Peptide Antagonizes Cell Activation by LPS—It was recently demonstrated that lipolan (Fig. 1) acts as an inhibitor of TLR2-mediated IL-8 secretion induced by the biologically active synthetic lipopeptide Pam3CSK4 (46Seyberth T. Voss S. Brock R. Wiesmueller K.H. Jung G. J. Med. Chem. 2006; 49: 1754-1765Crossref PubMed Scopus (26) Google Scholar). Because we did not observe any biological activity of lipolan in human mononuclear cells, we investigated the antagonistic activity of this TLR2 inhibitor with respect to stimulation by LPS (Fig. 3). Using human macrophages differentiated from peripheral blood mononuclear cells, we show here that lipolan is also able to antagonize cell activation by LPS in human macrophages, as shown by the reduction in TNFα production in the presence of lipolan. Antagonistic Activity of Lipolan Does Not Depend on TLR2—Cell activation by LPS depends on the presence of TLR4/MD-2, whereas lipopeptides induce cell activation via TLR2/TLR1 or TLR2/TLR6 heterodimer complexes (31Buwitt-Beckmann U. Heine H. Wiesmuller K.H. Jung G. Brock R. Akira S. Ulmer A.J. J. Biol. Chem. 2006; 281: 9049-9057Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). To get information concerning the involvement of TLRs in the antagonistic activity of lipolan against LPS, we used the defined HEK293 cell transient transfection system. HEK293 cells were transiently transfected with TLR4/MD-2 and the coreceptor CD14 and subsequently stimulated with LPS in t