Cell Activation of Human Macrophages by Lipoteichoic Acid Is Strongly Attenuated by Lipopolysaccharide-binding Protein

Abstract

Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor α under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor α and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung. Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor α under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor α and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung. The key to a successful pathogen defense is the recognition of pathogen-associated molecular structures by receptors of the innate immune system leading to a proinflammatory response. Systemic production of proinflammatory mediators, however, can also lead to sepsis, a complex clinical syndrome caused by an overshooting host response (1Cohen J. Nature. 2002; 420: 885-891Crossref PubMed Scopus (2157) Google Scholar). According to epidemiological studies, infections by Gram-positive bacteria are responsible for about half of the cases of systemic infections in the United States and Europe, and Staphylococcus aureus is the most frequently isolated Gram-positive pathogen in invasive infections and trauma patients (2Stillwell M. Caplan E.S. Infect. Dis. Clin. 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For Gram-negative bacteria, the pathogenesis of septic shock has been attributed to the activation of the innate immune system by lipopolysaccharide (LPS) 2The abbreviations used are: LPS, lipopolysaccharide; FRET, fluorescence resonance energy transfer; IL, interleukin; LBP, lipopolysaccharide-binding protein; LTA, lipoteichoic acid; PI-PLC, phosphatidylinositol-specific phospholipase C; PS, phosphatidylserine; TLR, toll-like receptor; TNF, tumor necrosis factor; sLBP, soluble LBP; mLBP, transmembrane configuration of LBP; NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE; Rh-PE, N-(rhodamine B sulfonyl)-PE; PE, phosphatidylethanolamine. 2The abbreviations used are: LPS, lipopolysaccharide; FRET, fluorescence resonance energy transfer; IL, interleukin; LBP, lipopolysaccharide-binding protein; LTA, lipoteichoic acid; PI-PLC, phosphatidylinositol-specific phospholipase C; PS, phosphatidylserine; TLR, toll-like receptor; TNF, tumor necrosis factor; sLBP, soluble LBP; mLBP, transmembrane configuration of LBP; NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE; Rh-PE, N-(rhodamine B sulfonyl)-PE; PE, phosphatidylethanolamine. from the outer leaflet of the outer membrane of the organisms (4Beutler B. 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Immun. 2001; 69: 6942-6950Crossref PubMed Scopus (166) Google Scholar, 22Mueller M. Lindner B. Kusumoto S. Fukase K. Schromm A.B. Seydel U. J. Biol. Chem. 2004; 279: 26307-26313Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Other reports show complex vectorial transport chains for LPS monomers (23Gioannini T.L. Teghanemt A. Zhang D. Coussens N.P. Dockstader W. Ramaswamy S. Weiss J.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4186-4191Crossref PubMed Scopus (296) Google Scholar). In contrast to the well investigated receptor system for LPS from Gram-negative bacteria, little is known about the molecular mechanisms involved in the recognition of Gram-positive pathogens. Several studies suggest that also molecules of the cell wall mediate inflammatory responses of the host, namely peptidoglycan of the murein layer and lipoteichoic acid (LTA). LTA is an amphiphilic molecule anchored to the outer surface of the cytoplasmic membrane by a glycerolipid. Peptidoglycan and LTA are released from the cell wall during growth and especially under antibiotic treatment, and both molecules have been shown to express immune stimulatory activity (24Gupta D. Kirkland T.N. Viriyakosol S. Dziarski R. J. Biol. Chem. 1996; 271: 23310-23316Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 25Morath S. Geyer A. Hartung T. J. Exp. Med. 2001; 193: 393-397Crossref PubMed Scopus (373) Google Scholar). After a long controversy about the immune stimulatory capacity and purity of commercial LTA preparations, establishment of a new purification protocol for LTA based on butanol extraction revealed that pure, LPS-free preparations of LTA from S. aureus exhibit immune stimulatory activity in human whole blood in high concentrations (25Morath S. Geyer A. Hartung T. J. Exp. Med. 2001; 193: 393-397Crossref PubMed Scopus (373) Google Scholar). LTA derived by this purification protocol was shown to be composed of a diacylglycerol lipid anchor covalently linked to a gentiobiose and a polymeric backbone that in S. aureus is composed of a central 1–3-linked glycerophosphate chain substituted with d-alanine, α-d-N-acetylglucosamine, and to a lesser extent unsubstituted 1–3-linked glycerophosphate residues. The immune stimulatory capacity of LTA could be confirmed by chemical synthesis of the lipid anchor containing a backbone of six glycerophosphate units carrying d-alanine and N-acetylglucosamine as substituents (26Morath S. Stadelmaier A. Geyer A. Schmidt R.R. Hartung T. J. Exp. Med. 2002; 195: 1635-1640Crossref PubMed Scopus (208) Google Scholar). However, the structural basis of cell activation by molecules derived from the Gram-positive cell wall is not conclusively defined. Although the molecular structures leading to activation of NOD proteins by peptidoglycan have recently been identified (27Girardin S.E. Boneca I.G. Carneiro L.A. Antignac A. Jehanno M. Viala J. Tedin K. Taha M.K. Labigne A. Zahringer U. Coyle A.J. DiStefano P.S. Bertin J. Sansonetti P.J. Philpott D.J. Science. 2003; 300: 1584-1587Crossref PubMed Scopus (1248) Google Scholar, 28Girardin S.E. Boneca I.G. Viala J. Chamaillard M. Labigne A. Thomas G. Philpott D.J. Sansonetti P.J. J. Biol. Chem. 2003; 278: 8869-8872Abstract Full Text Full Text PDF PubMed Scopus (1906) Google Scholar), the molecular basis of its TLR2 activity is not fully understood (29Travassos L.H. Girardin S.E. Philpott D.J. Blanot D. Nahori M.A. Werts C. Boneca I.G. EMBO Rep. 2004; 5: 1000-1006Crossref PubMed Scopus (392) Google Scholar, 30Dziarski R. Gupta D. Infect. Immun. 2005; 73: 5212-5216Crossref PubMed Scopus (194) Google Scholar). Also, recent data indicate that alanine substitution of LTA might not be required for its biological activity (31Henneke P. Morath S. Uematsu S. Weichert S. Pfitzenmaier M. Takeuchi O. Muller A. Poyart C. Akira S. Berner R. 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In the current study, we investigated the role of serum and LBP in cell activation by LTA. Our data show that LTA interacts with LBP and that cell activation is strongly attenuated by this interaction. We present evidence for a differential regulation of LTA recognition by macrophages dependent on the absence and presence of LBP. Reagents—Deep rough mutant LPS (Re LPS) was extracted from Salmonella enterica sv. Minnesota strain R595 according to the phenol/chloroform/petrol ether procedure (37Galanos C. Luderitz O. Rietschel E.T. Westphal O. Brade H. Brade L. Freudenberg M. Schade U. Imoto M. Yoshimura H. Kusumoto S. Shiba T. Eur. J. Biochem. 1985; 148: 1-5Crossref PubMed Scopus (401) 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. Highly purified lipoteichoic acid was isolated from S. aureus as described previously (25Morath S. Geyer A. Hartung T. J. Exp. Med. 2001; 193: 393-397Crossref PubMed Scopus (373) Google Scholar). LPS and LTA were suspended in phosphate-buffered saline (Biochrom, Berlin, Germany) by thorough vortexing. The suspensions were temperature cycled at least twice between 4 and 56 °C, with each cycle followed by intense vortexing for a few min, and then stored at 4 °C for at least 12 h prior to measurement. Suspensions were aliquoted and stored at –20 °C. Phosphatidylserine (PS) from bovine brain was purchased from Avanti-Polar Lipids (Alabaster, AL) and 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). The monoclonal mouse anti-mouse LBP antibody biG33 cross-reacting with human LBP and the monoclonal anti-human CD14 antibody biG 14 were obtained from Biometec (Greifswald, Germany). Isotype-matched IgG1 control antibody was obtained from BD Biosciences (Heidelberg, Germany). PI-PLC was from Sigma. Preparation of Macrophages and Incubation Conditions—Monocytes were isolated from human peripheral blood of healthy donors by the Hypaque-Ficoll gradient method and cultivated at 37 °C and 6% CO2 in Teflon bags in RPMI 1640 medium (endotoxin ≤0.01 EU/ml; Biochrom) containing 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, and 4% heat-inactivated human serum type AB from healthy donors. The 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, the cells were seeded at 200-μl aliquots of a suspension of 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. Cell-free supernatants were collected 4 h after stimulation for TNFα determination or 24 h after stimulation for IL-6 and IL-8 determination, respectively, and stored at –20 °C until determination of cytokine content. The data shown are the means and standard deviations (±S.D.) of triplicate samples of one experiment and representative of at least three independent experiments. Alveolar macrophages of the rat were isolated by lung lavage of male Sprague-Dawley rats (Charles River, Sulzfeld, Germany) as described (38Wu Y. Adam S. Hamann L. Heine H. Ulmer A.J. Buwitt-Beckmann U. Stamme C. Am. J. Respir. Cell Mol. Biol. 2004; 31: 587-594Crossref PubMed Scopus (34) Google Scholar). Cell viability was checked by erythrosin B exclusion and routinely averaged 94–98%. The cells were washed once in RPMI and resuspended in RPMI containing 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mm l-glutamine, with or without 10% heat-inactivated fetal calf serum (Linaris, Bettingen, Germany). To determine cytokine induction by LTA, the cells were seeded at 0.5 × 106 cells/well in 96-well tissue culture dishes and stimulated with LTA in the absence or presence of serum. To cleave cell-bound CD14 from the cell surface, the cells were treated with 0.5 unit/ml PI-PLC for 60 min at 37 °C prior to stimulation. Cell-free supernatants were harvested after 4 h of stimulation for the determination of TNFα. Cytokine Determination—Human TNFα and human IL-6 were determined in pooled cell-free supernatants of stimulated cells by sandwich enzyme-linked immunosorbent assay using monoclonal mouse antibody against human IL-6 and POD-conjugated rabbit anti-human IL-6 antibody and monoclonal mouse antibody against human TNFα and POD-conjugated rabbit anti-human TNFα antibody, respectively (Intex, Muttant, Switzerland) as stated in detail elsewhere (39Mueller M. Brandenburg K. Dedrick R. Schromm A.B. Seydel U. J. Immunol. 2005; 174: 1091-1096Crossref PubMed Scopus (62) Google Scholar). Rat TNFα and human IL-8 were determined in pooled cell-free supernatants of stimulated cells by sandwich enzyme-linked immunosorbent assay using Cytosets from BIOSOURCE (Solingen, Germany) exactly according to the manufacturer’s protocol. The data shown are the means ± 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 (17Schromm A.B. Brandenburg K. Rietschel E.T. Flad H.D. Carroll S.F. Seydel U. FEBS Lett. 1996; 399: 267-271Crossref PubMed Scopus (111) Google Scholar, 40Struck D.K. Hoekstra D. Pagano R.E. Biochemistry. 1981; 20: 4093-4099Crossref PubMed Scopus (1136) Google Scholar) to obtain information on the intercalation of LBP and LTA into liposomes made from the negatively charged phospholipid PS. For the FRET experiments, liposomes were double-labeled with NBD-PE and Rh-PE in chloroform [PS]:[NBD-PE]:[Rh-PE] of 100:1:1 molar ratios. The solvent was evaporated under a stream of nitrogen, and the lipids were resuspended in phosphate-buffered saline, 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, with each cycle followed by intense vortexing for a few min, 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). LBP (5,5 μg/ml) and LTA aggregates (30 μg/ml) were added to liposomes after 50 and 100 s, respectively. Because FRET spectroscopy is used here as a probe dilution assay, intercalation of unlabeled molecules such as LBP or LTA 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 a decrease of the acceptor intensities. For a qualitative analysis of experiments, the quotient 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. Plasmon Resonance Spectroscopy—A surface plasmon resonance technique (41Malmqvist M. Nature. 1993; 361: 186-187Crossref PubMed Scopus (525) Google Scholar) was used as a binding assay to detect interaction of LBP and LPS with immobilized liposomes made from PS. First, L1 sensor chip (Biacore AB, Uppsala, Sweden) was pretreated with a 10 μm suspension of PS liposomes to obtain an immobilized lipid matrix for interaction experiments with LBP and LTA. LBP and LTA were added at concentrations of 10 μm and 100 nm, respectively. The running buffer was phosphate-buffered saline at pH 7.0, and the experiments were performed at 37 °C at a flow rate of 10 μl/min in a BIACORE 3000. The data are presented as response units in dependence on time for one representative experiment of a total of three. Cell Activation of Human Macrophages by LTA Is Attenuated in the Presence of Serum—We tested the potential immune stimulatory activity of LPS-free LTA preparations using human macrophages that were in vitro differentiated from peripheral blood mononuclear cells. Stimulation of macrophages with LTA in the presence of 4% human serum led to a dose-dependent production of the proinflammatory cytokine TNFα (Fig. 1). Under these conditions, 3–10 μg/ml LTA were required to induce TNFα production. Compared with the amount of TNFα induced by the same cells after stimulation with 1 ng/ml LPS under serum-free conditions (1046 ± 24 pg TNFα/ml), the amounts of TNFα induced by these high concentrations of LTA were about three times lower (335 ± 7pgTNFα/ml induced by 10 μg/ml LTA). However, stimulation of the cells with LTA under serum-free culture conditions led to a significant enhancement of the cytokine production. Under these conditions, high concentrations of LTA induced similar amounts of TNFα (1002 ± 48 pg TNFα/ml induced by 1 μg/ml LTA) as induced by 1 ng LPS. In contrast to serum-containing conditions, TNFα production was already induced by 30 ng/ml LTA under serum-free conditions and increased dose-dependently with increasing concentrations of LTA, reaching saturation of cell activation at 300 ng/ml LTA. Similar results could be observed for the late pro-inflammatory mediators IL-6 and IL-8, both of which showed a dose-dependent inhibition in the presence of serum after 24 h of stimulation (Fig. 2). These results suggest that a compound in serum strongly attenuates cell activation by LTA.FIGURE 2Stimulation of late pro-inflammatory mediators by LTA in human macrophages is much more sensitive under serum-free conditions. Human blood macrophages were stimulated in the absence (left panel) or presence (right panel) of 4% AB serum with the indicated concentrations of LTA. Cell-free supernatants were harvested after 24 h for the determination of IL-6 (A and B) and IL-8 (C and D). Cytokine and chemokine concentrations are the means ± S.D. of triplicates. The data shown are representative of three independent experiments. N.D., not detectable.View Large Image Figure ViewerDownload Hi-res image Download (PPT) LTA Interacts with LBP and Inhibits the Integration of LBP into Phospholipid Membranes—LBP is an important serum protein involved in the innate immune recognition of a variety of pathogen-associated molecules. It has been shown to interact with LPS and enhance cell activation at low concentrations of LPS. However, at high concentrations of LBP as they appear in acute phase serum, inhibitory effects of LBP on cellular responses to LPS have been reported (21Gutsmann T. Mueller M. Carroll S.F. MacKenzie R.C. Wiese A. Seydel U. Infect. Immun. 2001; 69: 6942-6950Crossref PubMed Scopus (166) Google Scholar, 42Zweigner J. Gramm H.J. Singer O.C. Wegscheider K. Schumann R.R. Blood. 2001; 98: 3800-3808Crossref PubMed Scopus (176) Google Scholar, 43Hamann L. Alexander C. Stamme C. Za¨hringer U. Schumann R.R. Infect. Immun. 2005; 73: 193-200Crossref PubMed Scopus (56) Google Scholar). Because LBP has been found to interact also with other virulence factors (44Schroder N.W. Opitz B. Lamping N. Michelsen K.S. Za¨hringer U. Gobel U.B. Schumann R.R. J. Immunol. 2000; 165: 2683-2693Crossref PubMed Scopus (128) Google Scholar, 45Weber J.R. Freyer D. Alexander C. Schroder N.W. Reiss A. Kuster C. Pfeil D. Tuomanen E.I. Schumann R.R. Immunity. 2003; 19: 269-279Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 46Schroder N.W. Heine H. Alexander C. Manukyan M. Eckert J. Hamann L. Gobel U.B. Schumann R.R. J. Immunol. 2004; 173: 2683-2691Crossref PubMed Scopus (130) Google Scholar), we postulated that LBP may be the component in serum modulating cellular response to LTA. We used a liposome assay based on fluorescence resonance energy transfer to gain information on the interaction of LTA with LBP. We have shown in previous reports that LBP is not only a soluble protein but readily intercalates into liposomal membranes (20Gutsmann T. Haberer N. Carroll S.F. Seydel U. Wiese A. Biol. Chem. 2001; 382: 425-434Crossref PubMed Scopus (38) Google Scholar, 21Gutsmann T. Mueller M. Carroll S.F. MacKenzie R.C. Wiese A. Seydel U. Infect. Immun. 2001; 69: 6942-6950Crossref PubMed Scopus (166) Google Scholar). This hydrophobic interaction of LBP with phospholipid membranes is markedly enhanced for negatively charged phospholipids. In the presence of LPS, LBP mediates a transport of LPS into the phospholipid bilayer of liposomes (17Schromm A.B. Brandenburg K. Rietschel E.T. Flad H.D. Carroll S.F. Seydel U. FEBS Lett. 1996; 399: 267-271Crossref PubMed Scopus (111) Google Scholar). We used this liposome assay to investigate the influence of LTA on the interaction of LBP with liposomal membranes. When recombinant human LBP is added to liposomes composed of the negatively charged phospholipid phosphatidylserine, it readily intercalates into the phosholipid bilayer as can be taken from the increase in the FRET signal (ratio of donor and acceptor signals; Fig. 3). When LTA is added to the liposomes in the absence of LBP, no change in the FRET signal could be observed, indicating that LTA does not intercalate into the phospholipid membrane spontaneously. Additional application of LBP to the mixture of liposomes and LTA did not lead to any changes in the FRET signal. LTA Does Not Interact with Negatively Charged Phospholipid Membranes in the Absence or Presence of LBP—To further investigate the interaction of LTA and LBP with phospholipid membranes, we employed plasmon resonance spectroscopy. This method provides information on the binding of different partners in a flow-through system. We coated phosphatidylserine liposomes onto the surface of the flow-through chip and subsequently added LTA and LBP. As can be seen from Fig. 4, the addition of LTA did not lead to an increase of response units (time point 3), indicating that LTA did not bind to the phospholipid membranes. When LBP was added (time point 4), binding of LBP to the phospholipid membrane resulted in an increase in response units. Because the experiments were performed under continuous flow-through conditions, LTA was not present at the time of addition of LBP, explaining the absence of any inhibitory effects of LTA on the interaction of LBP with the membrane. In accordance to the results observed in the liposome assay, the addition of LTA to membrane-bound LBP (time points 6 and 7) did not lead to an increase of response units. These results confirm that LTA does not bind to phospholipid membranes in the absence or presence of membrane-associated LBP. In addition, there is no indication for any binding of LTA to membrane-associated LBP. LBP Inhibits LTA-induced Cytokin