Endotoxin (Lipopolysaccharide) Neutralization by Innate Immunity Host-Defense Peptides
Binding of lipopolysaccharide (LPS) to macrophages results in proinflammatory cytokine secretion. In extreme cases it leads to endotoxic shock. A few innate immunity antimicrobial peptides (AMPs) neutralize LPS activity. However, the underlying mechanism and properties of the peptides are not yet clear. Toward meeting this goal we investigated four AMPs and their fluorescently labeled analogs. These AMPs varied in composition, length, structure, and selectivity toward cells. The list included human LL-37 (37-mer), magainin (24-mer), a 15-mer amphipathic α-helix, and its d,l-amino acid structurally altered analog. The peptides were investigated for their ability to inhibit LPS-mediated cytokine release from RAW264.7 and bone marrow-derived primary macrophages, to bind LPS in solution, and when LPS is already bound to macrophages (fluorescence spectroscopy and confocal microscopy), to compete with LPS for its binding site on the CD14 receptor (flow cytometry) and affect LPS oligomerization. We conclude that a strong binding of a peptide to LPS aggregates accompanied by aggregate dissociation prevents LPS from binding to the carrier protein lipopolysaccharide-binding protein, or alternatively to its receptor, and hence inhibits cytokine secretion. Binding of lipopolysaccharide (LPS) to macrophages results in proinflammatory cytokine secretion. In extreme cases it leads to endotoxic shock. A few innate immunity antimicrobial peptides (AMPs) neutralize LPS activity. However, the underlying mechanism and properties of the peptides are not yet clear. Toward meeting this goal we investigated four AMPs and their fluorescently labeled analogs. These AMPs varied in composition, length, structure, and selectivity toward cells. The list included human LL-37 (37-mer), magainin (24-mer), a 15-mer amphipathic α-helix, and its d,l-amino acid structurally altered analog. The peptides were investigated for their ability to inhibit LPS-mediated cytokine release from RAW264.7 and bone marrow-derived primary macrophages, to bind LPS in solution, and when LPS is already bound to macrophages (fluorescence spectroscopy and confocal microscopy), to compete with LPS for its binding site on the CD14 receptor (flow cytometry) and affect LPS oligomerization. We conclude that a strong binding of a peptide to LPS aggregates accompanied by aggregate dissociation prevents LPS from binding to the carrier protein lipopolysaccharide-binding protein, or alternatively to its receptor, and hence inhibits cytokine secretion. Lipopolysaccharide (LPS), 2The abbreviations used are: LPSlipopolysaccharideAMPsantimicrobial peptidesLBPlipopolysaccharide-binding proteinRP-HPLCreverse-phase high performance liquid chromatographyNLLSQnonlinear least-squaresTNFtumor necrosis factorILinterleukinDMEMDulbecco's modified Eagle's mediumRTreverse transcriptionFITCfluorescein isothiocyanateNBD7-nitrobenz-2-oxa-1,3-diazole-4-ylMBHA4-methylbenzhydrylamine. 2The abbreviations used are: LPSlipopolysaccharideAMPsantimicrobial peptidesLBPlipopolysaccharide-binding proteinRP-HPLCreverse-phase high performance liquid chromatographyNLLSQnonlinear least-squaresTNFtumor necrosis factorILinterleukinDMEMDulbecco's modified Eagle's mediumRTreverse transcriptionFITCfluorescein isothiocyanateNBD7-nitrobenz-2-oxa-1,3-diazole-4-ylMBHA4-methylbenzhydrylamine. also termed endotoxin, is an integral structural component of the outer membrane of Gram-negative bacteria (1Raetz C.R. Whitfield C. Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3222) Google Scholar). LPS is released from the bacteria during cell division, cell death, or in particular, as a result of antibiotic treatment against bacterial infection (2Hopkin D.A. Lancet. 1978; 2: 1193-1194PubMed Google Scholar, 3Hancock R.E. Scott M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8856-8861Crossref PubMed Scopus (809) Google Scholar). Upon its release, LPS is recognized by mononuclear phagocytes (monocytes and macrophages), which are part of the innate immunity of the host, and activates them. This results in an increase in their phagocytic activity and significantly enhances the secretion of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and others (4Rietschel E.T. Brade H. Holst O. Brade L. Muller-Loennies S. Mamat U. Zahringer U. Beckmann F. Seydel U. Brandenburg K. Ulmer A.J. Mattern T. Heine H. Schletter J. Loppnow H. Schonbeck U. Flad H.D. Hauschildt S. Schade U.F. 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The activation mechanism of macrophages by LPS starts when LPS (through its toxic entity, lipid A) binds with LPS-binding protein (LBP), accelerating the binding of LPS to CD14, the primary receptor of LPS, which is expressed mainly on macrophages (9Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3351) Google Scholar, 10Schumann R.R. Leong S.R. Flaggs G.W. Gray P.W. Wright S.D. Mathison J.C. Tobias P.S. Ulevitch R.J. Science. 1990; 249: 1429-1431Crossref PubMed Scopus (1363) Google Scholar, 11Tobias P.S. Ulevitch R.J. Immunobiology. 1993; 187: 227-232Crossref PubMed Scopus (150) Google Scholar). The LPS-CD14 complex initiates intracellular signaling by interacting with the transmembrane protein Toll-like receptor-4 (TLR-4), which activates the NF-κB transcription factor, resulting in the production and secretion of pro-inflammatory cytokines (12Hailman E. Lichenstein H.S. Wurfel M.M. Miller D.S. Johnson D.A. Kelley M. 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Immunol. 2000; 165: 3541-3544Crossref PubMed Scopus (274) Google Scholar). lipopolysaccharide antimicrobial peptides lipopolysaccharide-binding protein reverse-phase high performance liquid chromatography nonlinear least-squares tumor necrosis factor interleukin Dulbecco's modified Eagle's medium reverse transcription fluorescein isothiocyanate 7-nitrobenz-2-oxa-1,3-diazole-4-yl 4-methylbenzhydrylamine. lipopolysaccharide antimicrobial peptides lipopolysaccharide-binding protein reverse-phase high performance liquid chromatography nonlinear least-squares tumor necrosis factor interleukin Dulbecco's modified Eagle's medium reverse transcription fluorescein isothiocyanate 7-nitrobenz-2-oxa-1,3-diazole-4-yl 4-methylbenzhydrylamine. In an attempt to understand the mechanism of macrophage stimulation by LPS, two major approaches have been reported. The first one utilized LPS receptor antagonists including anti-CD14 antibodies, anti-LBP antibodies, and lipid A analogs, all of which bind to essential components participating in the signaling mechanism (17Schimke J. Mathison J. Morgiewicz J. Ulevitch R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13875-13880Crossref PubMed Scopus (127) Google Scholar, 18Stover A.G. Da Silva Correia J. Evans J.T. Cluff C.W. Elliott M.W. Jeffery E.W. Johnson D.A. Lacy M.J. Baldridge J.R. Probst P. Ulevitch R.J. Persing D.H. Hershberg R.M. J. Biol. Chem. 2004; 279: 4440-4449Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The second approach utilized LPS blockers such as anti-lipid A antibodies and modified liposomes, both of which bind to LPS itself and prevent its ability to activate the macrophages (19David S.A. J. Mol. Recognit. 2001; 14: 370-387Crossref PubMed Scopus (102) Google Scholar, 20Manocha S. Feinstein D. Kumar A. Expert Opin. Investig. Drugs. 2002; 11: 1795-1812Crossref PubMed Scopus (47) Google Scholar). Note, however, that although these studies have helped us to understand the steps involved in LPS neutralization, the approaches used could not deal with the bacteria from which the LPS is derived (19David S.A. J. Mol. Recognit. 2001; 14: 370-387Crossref PubMed Scopus (102) Google Scholar). Recent studies have shown that a few antimicrobial peptides (AMPs) also have the potential to neutralize LPS-induced endotoxic effects. These peptides are important components of the innate defense system of all species of life (21Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1492) Google Scholar). They are produced in large quantities at the site of infection and/or inflammation and act rapidly to clear microbes (22Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6575) Google Scholar). Several AMPs prevent LPS-dependent cytokine induction in macrophages and block sepsis in animal models (23Larrick J.W. Hirata M. Balint R.F. Lee J. Zhong J. Wright S.C. Infect. Immun. 1995; 63: 1291-1297Crossref PubMed Google Scholar, 24Hirata M. Zhong J. Wright S.C. Larrick J.W. Prog. Clin. Biol. Res. 1995; 392: 317-326PubMed Google Scholar, 25Gough M. Hancock R.E. Kelly N.M. Infect. Immun. 1996; 64: 4922-4927Crossref PubMed Google Scholar, 26Scott M.G. Yan H. Hancock R.E. Infect. Immun. 1999; 67: 2005-2009Crossref PubMed Google Scholar, 27Giacometti A. Cirioni O. Ghiselli R. Mocchegiani F. Del Prete M.S. Viticchi C. Kamysz W. Lempicka E. Saba V. Scalise G. Antimicrob. Agents Chemother. 2002; 46: 2132-2136Crossref PubMed Scopus (55) Google Scholar). These studies also showed a direct correlation between the ability of AMPs to bind LPS and their antimicrobial activity. Furthermore, it has been shown that other factors are involved, such as peptide hydrophobicity and amphipathicity and a defined structure (28David S.A. Awasthi S.K. Balaram P. J. Endotoxin Res. 2000; 6: 249-256Crossref PubMed Scopus (26) Google Scholar, 29Scott M.G. Vreugdenhil A.C. Buurman W.A. Hancock R.E. Gold M.R. J. Immunol. 2000; 164: 549-553Crossref PubMed Scopus (246) Google Scholar, 30Nagaoka I. Hirota S. Niyonsaba F. Hirata M. Adachi Y. Tamura H. Tanaka S. Heumann D. Clin. Diagn. Lab. Immunol. 2002; 9: 972-982PubMed Google Scholar). To better understand the mechanism by which AMPs block LPS-dependent cytokine induction and the peptide properties required for this activity, we investigated four AMPs and their fluorescently labeled analogs. The peptides vary in their amino acid composition, length, structure, and selectivity toward different cells. The list includes two native antimicrobial peptides, human LL-37 (37-mer) and magainin (24-mer), as well as a 15-mer amphipathic α-helical peptide composed of Leu and Lys, and its d,l-amino acid diastereomer, a structurally altered analog. The peptides were investigated for their ability to inhibit LPS-induced cytokine release and to bind LPS in solution and when bound to macrophages and affect its oligomerization, as well as to compete with LPS on its binding site on the CD14 receptor and LBP. The results are discussed in view of the essential properties of a peptide required for LPS as well as of and amino were from used for peptide included acid and Dulbecco's modified Eagle's medium and amino acid solution were by from and for for lipopolysaccharide from and fluorescein isothiocyanate lipopolysaccharide were by by reverse from and peptides were by a on by an peptide of the of the peptides with and 7-nitrobenz-2-oxa-1,3-diazole-4-yl on the peptide as Y. Y. 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The peptides were from the by with and with acid of the peptides bound to which in peptide one major as by that by The peptides were by on a reverse-phase The in by a of in both acid at a of The peptides were shown to by The peptides were to amino acid and spectroscopy to their and of the activity of the peptides in in a of as of a bacteria at a of in medium were to of the peptide in in of by the at with a an of at were expressed as the the at which of of of Gram-negative bacteria were E. and of bone macrophages were with from the and of The were from the with the were to a of in medium with from the medium of that and were in bacterial for at were by the and were in macrophages were in with and at in a at and were a A used for this well cell in medium with RAW264.7 macrophage in the first and second as and and medium Dulbecco's to the first and second and of peptides from the solution in were to the for of peptide The for to well of solution acid and in a of The at in an a of the with and to the and results were The results were in at The for peptide from the of cell the of peptide and from the at which cell The of AMPs on Binding to RAW264.7 were for at with in the or of the peptides and in with were with and the peptides were for and the the binding of to RAW264.7 by of on LPS-dependent were in and were or with LPS in the or of peptide in with for the were from the and with to the The and of the were The were used as a in The were in the of and and and A and in to the and to the of The steps for the of a at by or of at for at or and for and an at for the the at for and to The were and on The of the were by a system of from RAW264.7 and macrophages were in The medium by the to well of with of The were with LPS in the of and or In bone marrow-derived macrophages were with LPS in the of and and that were with LPS and as The were for at which of the medium from treatment were in the a to the were in Binding were as D. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). LPS of solution to peptide and all in The in were as a of the a with at the system To for the the of both and LPS at the The in the the of LPS bound to the is to its in a D. Y. J. Biol. Chem. Full Text PDF PubMed Google us to the LPS binding to the system binding at a and the could from the between the of peptide and the LPS a The were by nonlinear least-squares and The the is the LPS is the in the of peptide and the of LPS the LPS bound or the and is the In the were against the of of LPS and the of the bound to of on as P.S. K. D. Ulevitch R.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). with of the The in the of were by a with at The of both and the peptides were as a of the aggregates results in an increase in the of of C.J. J. J. Immunol. 2000; PubMed Scopus Google Scholar). The in were the system and of and LPS Binding to were in an system and The were and for at with in with the were and the with peptides and that were with or with peptides were used as The were and with The and of the peptides and on the confocal and AMPs were and The list includes human a 15-mer peptide composed of and and its d,l-amino acid The 15-mer to an amphipathic structure as in a and structure M. J. Full Text PDF PubMed Scopus Google Scholar). The that all AMPs are against both the and Gram-negative bacteria The fluorescently labeled peptides the activity of the peptides not The peptides were not toxic to macrophages at the in the LPS and of the peptides and amino are of the peptides are in their and amino are of the peptides are in their in a properties of is in binding on LPS on the increase in the of treatment with the AMPs LL-37 and by LPS LPS is in The on the increase in the of treatment with the AMPs LL-37 and in a of the Binding of to RAW264.7 macrophages were with in the or of as a The results shown in and in and that and inhibit binding to the macrophages in a magainin not significantly inhibit binding to the cells. the peptide and the binding of LPS by and at and at of LPS-mediated macrophages were with LPS with or the peptides as a for to the of the peptides on and the The shown in that which all other peptides were showed that and LL-37 a in at the it to the These results with the of the peptides to inhibit the binding of LPS to macrophages not with their antimicrobial activity, their to an α-helical structure, or the of their binding to LPS discussed in the that in different of were used on the of these However, of binding to LPS for all the peptides is at an on the binding of the peptides to all the were of a large of peptides with of used a to in the cell medium stimulation with LPS and for RAW264.7 and bone primary macrophages, in the of different of a which to in the The peptides at a of the to the the with the magainin on of secretion from bone primary macrophages not to the to that of the that the activity of in this is in RAW264.7 This is in with the LPS required to the primary macrophages with Binding of to LPS in of the by which antimicrobial peptides are to neutralize LPS is by binding to LPS, its binding to M.G. Vreugdenhil A.C. Buurman W.A. Hancock R.E. Gold M.R. J. Immunol. 2000; 164: 549-553Crossref PubMed Scopus (246) Google Scholar). We binding by peptides as D. Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Biochem. J. 1990; PubMed Scopus Google Scholar). The of the peptides as a of LPS the shown in to the in which is in The in the the of peptide bound to LPS, the hydrophobicity of its The were by and were to high and for all of the peptides the that magainin is These were to the that were from the These that the activity of a peptide is not to their ability to bind Binding of to in the or of the binding of peptides to macrophages or their with by confocal of the macrophages with labeled peptides that and LL-37 bound to the better and and the peptides could also the This is in with their in However, when peptides were with the macrophages binding and all of the peptides in bound and with LPS in not to the cell peptides were with LPS the cell as LPS is to with CD14 binding J. 1995; Google Scholar). These results are in with the high of the peptides to LPS which is their to the cell Y. PubMed Scopus Google Scholar). the of these we the that binding to macrophage membrane or to LPS is a mechanism of LPS The of to LPS to has been shown to compete with LPS binding to its primary receptor CD14 I. Hirota S. Niyonsaba F. Hirata M. Adachi Y. Tamura H. Tanaka S. Heumann D. Clin. Diagn. Lab. Immunol. 2002; 9: 972-982PubMed Google Scholar, I. Hirota S. Niyonsaba F. Hirata M. Adachi Y. Tamura H. Heumann D. J. Immunol. 2001; PubMed Scopus Google Scholar). To this is by all peptides, we the macrophages with and the macrophages with the The shown in that of the peptides LL-37 could significantly LPS its binding to These a different of for the other peptides, which not receptor This is in with studies that LL-37 LPS by binding to LPS and also by with it on its receptor, CD14 I. Hirota S. Niyonsaba F. Hirata M. Adachi Y. Tamura H. Tanaka S. Heumann D. Clin. Diagn. Lab. Immunol. 2002; 9: 972-982PubMed Google Scholar). of on aggregates in K. D.H. P. C. J. Biol. Chem. 1994; Full Text PDF PubMed Google which are to by their to CD14 P.S. Ulevitch R.J. Immunobiology. 1993; 187: 227-232Crossref PubMed Scopus (150) Google Scholar). We the potential of the peptides to aggregates and this with LPS The of aggregates is increase when the aggregates of The of the peptides on is shown in The that all the peptides other magainin have a strong on the of the of their is LL-37 This is in with their LPS activity are of effects. with and is an or The peptides a for and for This is in with the that is already at is at with LL-37 is a increase in the to the in the of magainin is a increase in the at which as the the for which is not clear. We investigated the correlation between the antimicrobial activity of AMPs and their ability to neutralize LPS, as well as the of LPS The important is that a strong binding of a peptide to LPS aggregates accompanied by aggregate dissociation prevents LPS from binding to the carrier protein or alternatively to its receptor, and hence inhibits cytokine secretion. This is on the of four antimicrobial peptides, all of which have antimicrobial activity in composition, structure, and ability to neutralize LPS and To prevent macrophage activation by LPS, a peptide to prevent the binding of LPS to the CD14 receptor M.A. Vogel S.N. Microbes Infect. 2002; 4: 903-914Crossref PubMed Scopus (432) Google Scholar, 14Woltmann A. Hamann L. Ulmer A.J. Gerdes J. Bruch H.P. Rietschel E.T. Langenbecks Arch. Surg. 1998; 383: 2-10Crossref PubMed Scopus (29) Google Scholar, C. Rietschel E.T. J. Endotoxin Res. 2001; Google Scholar). we first by that all of the peptides magainin block from binding to macrophages These with the of the peptides to the of of the two cytokines and as well as to prevent secretion from both RAW264.7 and primary macrophages with LPS in or to binding that all the peptides bind LPS in solution with high and and with a of between LPS and peptide Furthermore, confocal that all the peptides bind to LPS when it is already to the macrophages, on the membrane or when However, when to macrophages LL-37 and are bound in with the hydrophobicity of the peptides These not with the with the antimicrobial of the peptides and We conclude that strong binding of a peptide to LPS is not to block LPS activity. that Scott M.G. Yan H. Hancock R.E. Infect. Immun. 1999; 67: 2005-2009Crossref PubMed Google a correlation between the ability of a of peptides to bind E. LPS and their antimicrobial activity and ability to block and between the and LPS on the Binding the CD14 that LL-37 with which is already bound to its receptor that this peptide and LPS a binding site Note, that LL-37 not LPS from its receptor, that this mechanism is not the mechanism of LPS by These results that this is and is not by all In of this I. Hirota S. Niyonsaba F. Hirata M. Adachi Y. Tamura H. Tanaka S. Heumann D. Clin. Diagn. Lab. Immunol. 2002; 9: 972-982PubMed Google Scholar, I. Hirota S. Niyonsaba F. Hirata M. Adachi Y. Tamura H. Heumann D. J. Immunol. 2001; PubMed Scopus Google that and peptides, which to the bind to the cell CD14 and inhibit the binding of to the cells. between the and LPS on the Binding site on the mechanism by which AMPs inhibit LPS-induced release of cytokines have that direct binding of the peptides to LPS it to of the peptides with itself M.G. Vreugdenhil A.C. Buurman W.A. Hancock R.E. Gold M.R. J. 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Agents Chemother. 2004; PubMed Scopus Google Scholar). we that the peptides not bind as by the of their fluorescently labeled with to on not of LPS by the that the peptides and could LPS aggregates In the binding a between LPS and the peptide bound and LL-37 are the peptides by and the different LL-37 a and a and magainin a activity at a of The properties of the peptides, as in that the ability of AMPs to bind to LPS and to antimicrobial activity are not to neutralize LPS-induced macrophage To the peptides also to the LPS However, AMPs in compete with LPS on its binding site the CD14 receptor, as in the of the a peptide is major structural by the of not this the that antimicrobial peptides are in LPS, with studies the of such for in Y. PubMed Scopus Google Scholar, U. H.G. Y. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google potential to bacteria and at the