Monomeric endotoxin:protein complexes are essential for TLR4-dependent cell activation

Abstract

Potent TLR4-dependent cell activation by Gram-negative bacterial endotoxin depends on sequential endotoxin—protein and protein—protein interactions with LBP, CD14, MD-2 and TLR4. LBP and CD14 combine, in an albumin-dependent fashion, to extract single endotoxin molecules from purified endotoxin aggregates (E_agg) or the bacterial outer membrane and form monomeric endotoxin:CD14 complexes that are the preferred presentation of endotoxin for transfer to MD-2. Endotoxin in endotoxin:CD14 is readily transferred to MD-2, again in an albumin-dependent manner, to form monomeric endotoxin:MD-2 complex. This monomeric endotoxin:protein complex (endotoxin:MD-2) activates TLR4 at picomolar concentrations, independently of albumin, and is, therefore, the apparent ligand in endotoxin-dependent TLR4 activation. Tetra-, penta-, and hexa-acylated forms of meningococcal endotoxin (LOS) react similarly with LBP, CD14, and MD-2 to form endotoxin:MD-2 complexes. However, tetra- and penta-acylated LOS:MD-2 complexes are less potent TLR4 agonists than hexa-acylated LOS:MD-2. This is mirrored in the reduced activity of tetra-, penta- versus hexa-acylated LOS aggregates (LOS_agg) + LBP toward cells containing mCD14, MD-2, and TLR4. Therefore, changes in agonist potency of under-acylated meninigococcal LOS are determined by differences in properties of monomeric endotoxin:MD-2.

Keywords

CD14 Gram-negative bacteria lipopolysaccharide Toll-like receptors MD-2 lipopolysaccharide binding protein

References

Alexander C. , Rietschel ET Bacterial lipopolysaccharides and innate immunity . J Endotoxin Res 2001; 7: 167—202.

Heumann D. , Roger T. Initial responses to endotoxins and Gram-negative bacteria. Clin Chim Acta 2002; 323: 59—72.

Beutler B. TLR4 as the mammalian endotoxin sensor. Curr Top Microbiol Immunol 2002; 270: 109—120.

Ulevitch RJ , Tobias PS Recognition of Gram-negative bacteria and endotoxin by the innate immune system . Curr Opin Immunol 1999; 11: 19—22.

Miyake K. , Henneke P. , Golenbock DT Endotoxin recognition molecules, Toll-like receptor 4—MD-2. Innate immune recognition of lipopolysaccharide by endothelial cells. Semin Immunol 2004; 16: 11—16.

Giardina PC , Gioannini T. , Buscher BA et al. Construction of acetate auxotrophs of Neisseria meningitidis to study host-meningococcal endotoxin interactions. J Biol Chem 2001; 276: 5883—5891.

Gioannini TL , Zhang D. , Teghanemt A. , Weiss JP An essential role for albumin in the interaction of endotoxin with lipopolysaccharide-binding protein and sCD14 and resultant cell activation. J Biol Chem 2002; 277: 47818—47825.

Gioannini TL , Teghanemt A. , Zhang D. et al. Isolation of an endotoxin—MD-2 complex that produces Toll-like receptor 4-dependent cell activation at picomolar concentrations. Proc Natl Acad Sci USA 2004 ; 101: 4186—4191

Post DM , Ketterer MR , Phillips NJ , Gibson BW , Apicella MA The msbB mutant of Neisseria meningitidis strain NMB has a defect in lipooligosaccharide assembly and transport to the outer membrane. Infect Immun 2003; 71: 647—655.

10.

Kitchens RL , Ulevitch RJ , Munford RS Lipopolysaccharide (LPS) partial structures inhibit responses to LPS in a human macrophage cell line without inhibiting LPS uptake by a CD14mediated pathway. J Exp Med 1992; 176: 485—494.

11.

Yang H. , Young DW , Gusovsky F. , Chow JC

Cellular events mediated by lipopolysaccharide-stimulated Toll-like receptor 4. MD-2 is required for activation of mitogen-activated protein kinases and Elk-1. J

Biol Chem 2000; 275: 20861—20866.

12.

Tapping RI , Tobias PS Cellular binding of soluble CD14 requires lipopolysaccharide (LPS) and LPS-binding protein. J Biol Chem 1997; 272: 23157—23164.

13.

Gioannini TL , Teghanemt A. , Zarember KA , Weiss JP Regulation of interactions of endotoxin with host cells. J Endotoxin Res 2003; 9: 401—408.

14.

Iovine N. , Eastvold J. , Elsbach P. , Weiss JP , Gioannini TL The carboxyl-terminal domain of closely related endotoxin-binding proteins determines the target of protein-lipopolysaccharide complexes. J Biol Chem 2002; 277: 7970—7978.

15.

Kitchens RL , Munford RS CD14-dependent internalization of bacterial lipopolysaccharide (LPS) is strongly influenced by LPS aggregation but not by cellular responses to LPS. J Immunol 1998 ; 160: 1920—1928.

16.

da Silva Correia J. , Soldau K. , Christen U. , Tobias PS , Ulevitch RJ Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex: transfer from CD14 to TLR4 and MD-2. J Biol Chem 2001; 276: 1129—1135.

17.

Akashi S. , Saitoh S. , Wakabayashi Y. et al. Lipopolysaccharide interaction with cell surface Toll-like receptor 4—MD-2: higher affinity than that with MD-2 or CD14. J Exp Med 2003; 198: 1035—1042.

18.

Viriyakosol S. , Tobias PS , Kitchens RL , Kirkland TN MD-2 binds to bacterial lipopolysaccharide . J Biol Chem 2001; 276: 38044—38051.

19.

Visintin A. , Mazzoni A. , Spitzer JA , Segal DM Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4. Proc Natl Acad Sci USA 2001; 98: 12156—12161.

20.

Kennedy MN , Mullen GE , Leifer CA et al. A complex of soluble MD-2 and lipopolysaccharide serves as an activating ligand for Toll-like receptor 4. J Biol Chem 2004; 279: 34698—34704.

21.

Brandenburg K. , Wiese A. Endotoxins: relationships between structure, function, and activity. Curr Top Med Chem 2004; 4: 1127—1146.

22.

Darveau RP Lipid A diversity and the innate host response to bacterial infection. Curr Opin Microbiol 1998; 1: 36—42.

23.

Caroff M. , Karibian D. , Cavaillon JM , Haeffner-Cavaillon N. Structural and functional analyses of bacterial lipopolysaccharides. Microbes Infect 2002; 4: 915—926.

24.

Kitchens RL , Munford RS Enzymatically deacylated lipopolysaccharide (LPS) can antagonize LPS at multiple sites in the LPS recognition pathway . J Biol Chem 1995; 270: 9904—9910.

25.

Munford RS , Erwin AL Eukaryotic lipopolysaccharide deacylating enzyme. Methods Enzymol 1992; 209: 485—492.

26.

Somerville Jr JE , Cassiano L. , Bainbridge B. , Cunningham MD , Darveau RP A novel Escherichia coli lipid A mutant that produces an anti-inflammatory lipopolysaccharide. J Clin Invest 1996; 97: 359—365.

27.

van der Ley P. , Steeghs L. , Hamstra HJ , ten Hove J. , Zomer B. , van Alphen L. Modification of lipid A biosynthesis in Neisseria meningitidis lpxL mutants: influence on lipopolysaccharide structure, toxicity, and adjuvant activity. Infect Immun 2001; 69: 5981—5990.