A biophysical approach towards an understanding of endotoxin-induced signal transduction

Abstract

The purpose of our studies is to define the physicochemical parameters involved in the activation of host cells by endotoxin and to characterize the processes operative during endotoxin/membrane interaction with the aim of understanding transmembrane signal transduction mechanisms. To this end, we determined the molecular conformation of the lipid A component of various endotoxins (endotoxic conformation) using X-ray small angle diffraction, their intercalation into reconstituted macrophage membranes with fluorescence resonance energy transfer spectroscopy, and their IL-6 inducing capacity in whole blood. We also investigated their influence on ion channels as a possible primary event in cell activation applying patch-clamp techniques to macrophages. We found a strong influence of the molecular charge on the molecular conformation, and we could show that the presence of charged groups and a cone- or wedge-like molecular conformation of lipid A are prerequisites for the expression of bioactivity. We also obtained strong evidence supporting the idea that the interaction of endotoxin with ion channels is one of the very early events in the interaction with the cell and, most likely, in signal transduction.

References

Rietschel ET , Brade H. , Holst O. et al. Bacterial endotoxin: Chemical constitution, biological recognition, host response, and immunological detoxification. Curr Top Microbiol Immunol 1996; 216: 39—81.

Rietschel ET , Kirikae T. , Schade FU et al. Bacterial endotoxin: Molecular relationships of structure to activity and function. FASEB J 1994; 8: 217—225.

Brandenburg K. , Mayer H. , Koch Mhj , Weckesser J. , Rietschel ET , Seydel U. Influence of the supramolecular structure of free lipid A on its biological activity. Eur J Biochem 1993; 218: 555—563.

Seydel U. , Brandenburg K. , Rietschel ET A case for an endotoxic conformation. Prog Clin Biol Res 1994; 388: 17—30.

Wright SD , Ramos RA , Tobias PS , Ulevitch RJ , Mathison JC CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990; 249: 1431—1433.

Schumann RR , Leong SR , Flaggs GW et al. Structure and function of lipopolysaccharide binding protein . Science 1990; 249: 1429—1431.

Hailman E. , Lichenstein HS , Wurfel MM et al. Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J Exp Med 1994; 179 :269—277.

Yu B. , Wright SD Catalytic properties of lipopolysaccharide (LPS) binding protein — transfer of LPS to soluble CD14. J Biol Chem 1996; 271: 4100—4105.

Wurfel MM , Hailman E. , Wright SD Soluble CD14 acts as a shuttle in the neutralization of lipopolysaccharide (LPS) by LPS-binding protein and reconstituted high density lipoprotein. J Exp Med 1995; 181: 1743—1754.

10.

Schromm AB , Brandenburg K. , Rietschel ET , Flad H-D. , Carroll SF , Seydel U. Lipopolysaccharide binding protein (LBP) mediates CD14-independent intercalation of lipopolysaccharide into phospholipid membranes. FEBS Lett 1996; 399: 267—271.

11.

Yu B. , Hailman E. , Wright SD Lipopolysaccharide binding protein and soluble CD14 catalyze exchange of phospholipids . J Clin Invest 1997; 99: 315—324.

12.

Wurfel MM , Wright SD Lipopolysaccharide-binding protein and soluble CD14 transfer lipopolysaccharide to phospholipid bilayers - Preferential interaction with particular classes of lipid. J Immunol 1997; 158: 3925—3934.

13.

Flegel WA , Wölpl A. , Männel DN , Northoff H. Inhibition of endotoxin-induced activation of human monocytes by human lipoproteins . Infect Immun 1989; 57: 2237—2245.

14.

Thiéblemont N. , Wright SD Mice genetically hyporesponsive to lipopolysaccharide (LPS) exhibit a defect in endocytic uptake of LPS and ceramide. J Exp Med 1997; 185: 2095—2100.

15.

Dijkstra J. , Bron R. , Wilschut J. , de Haan A. , Ryan JL Activation of murine lymphocytes by lipopolysaccharide incorporated in fusogenic, reconstituted influenza virus envelopes (virosomes) . J Immunol 1996; 157: 1028—1036.

16.

Schromm AB , Brandenburg K. , Loppnow H. et al. The charge of endotoxin molecules influences their conformation and interleukin-6 inducing capacity. J Immunol 1998; 161: 5464—5471.

17.

Perera PY , Vogel SN , Detore GR , Haziot A. , Goyert SM CD14-dependent and CD14-independent signaling pathways in murine macrophages from normal and CD14 knockout mice stimulated with lipopolysaccharide or taxol . J Immunol 1997; 158: 4422—4429.

18.

Ingalls RR , Golenbock DT CD11c/CD18, a transmembrane signaling receptor for lipopolysaccharide. J Exp Med 1995; 181: 1473—1479.

19.

El-Samalouti VT , J Schletter , Brade H. et al. Detection of lipopolysaccharide (LPS)-binding membrane proteins by immunocoprecipitation with LPS and anti-LPS antibodies . Eur J Biochem 1997; 250: 418—424.

20.

El-Samalouti VT , Schletter J. , Chyla I. et al. Identification of the 80 kDa LPS-binding protein (LMP80) as decay accelerating factor (DAF, CD55). FEMS Immunol Microbiol 1999; 23: 259—269.

21.

Hu Y. , Fisette PL , Denlinger LC et al. Purinergic receptor modulation of lipopolysaccharide signaling and inducible nitric-oxide synthase expression in RAW 264.7 macrophages. J Biol Chem 1998; 273: 27170—27175.

22.

Ingalls RR , Arnaout MA , Golenbock DT Outside-in signaling by lipopolysaccharide through a tailless integrin. J Immunol 1997; 159: 433—438.

23.

Yang RB , Mark MR , Gray A. et al Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signaling. Nature 1998; 395: 284—288.