Immunostimulatory, but not antiendotoxin, activity of lipid X is due to small amounts of contaminating N,O-acylated disaccharide-1-phosphate: in vitro and in vivo reevaluation of the biological activity of synthetic lipid X.
Lipid X, a precursor in the biosynthesis of lipid A, has been claimed to possess most of the immunostimulatory activity but none of the toxicity of endotoxin. However, recent work shows that synthetic lipid X can be contaminated with small amounts of N,O-acylated disaccharide-1-phosphate (H. Aschauer, A. Grob, J. Hildebrandt, E. Schuetze, and P. Steutz, J. Biol. Chem. 265:9159-9164, 1990). Because the impurities themselves exhibit immunostimulatory properties, it was necessary to establish whether chemically pure synthetic lipid X exhibits any of the endotoxinlike biological activities previously attributed to the native compound extracted from the Escherichia coli MN7 mutant. In the present study, two batches of synthetic lipid X were used: batch A contained the contaminating disaccharide, and batch B was pure lipid X. Batch A, previously believed to be pure on the basis of spectroscopic and microanalysis data, induced murine peritoneal macrophages to secrete tumor necrosis factor, interleukin-1, and prostaglandin E2 at a minimal dose of 10 micrograms/ml in vitro. Batch B, further purified by Sephadex LH 20 chromatography, was found virtually inactive in these in vitro assays. Furthermore, batch A was pyrogenic in rabbits at a dose of 0.05 mg/kg, whereas batch B was not pyrogenic at doses of up to greater than or equal to 2 mg/kg. However, both batches were equally tolerated by galactosamine-loaded mice at doses of up to 100 mg/kg. Surprisingly, while only batch A protected neutropenic mice against lethal infection with Pseudomonas aeruginosa (50% effective dose, 12.4 mg/kg), both batches were equally protective against infection with herpes simplex virus type 1 in mice and guinea pigs, even when lipid X was administered therapeutically. Interestingly, both batches of lipid X blocked endotoxin-induced expression of monocyte procoagulant activity and priming of human neutrophils for superoxide anion release. Similarly, both batches protected galactosamine-sensitized mice from otherwise lethal endotoxemia when administered prophylactically or simultaneously with the lipopolysaccharide challenge. Thus, our findings suggest that chemically pure lipid X (batch B) is devoid of the immunostimulatory properties of lipid A or endotoxin. Rather, it behaves as a competitive inhibitor of lipopolysaccharide. We conclude, therefore, that previous studies which attributed immunostimulatory activities to any batch of synthetic lipid X should be interpreted with caution.
Lam
C
C
Sandoz Forschungsinstitut, Vienna, Austria.
Hildebrandt
J
J
Schütze
E
E
Rosenwirth
B
B
Proctor
R A
RA
Liehl
E
E
Stütz
P
P
D000276:Adjuvants, Immunologic; D000818:Animals; D000998:Antiviral Agents; D015232:Dinoprostone / Q000096:biosynthesis; D004187:Disaccharides / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D004731:Endotoxins / Q000037:antagonists & inhibitors*; D005260:Female; D005688:Galactosamine / Q000633:toxicity; D006017:Glycolipids / Q000138:chemical synthesis / Q000276:immunology* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000096:biosynthesis; D008033:Limulus Test; D008264:Macrophages / Q000502:physiology; D051379:Mice; D008815:Mice, Inbred Strains; D009000:Monocytes / Q000502:physiology; D009504:Neutrophils / Q000502:physiology; D011749:Pyrogens; D011817:Rabbits; D012790:Shwartzman Phenomenon; D013403:Sugar Phosphates / Q000276:immunology; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D000276:Adjuvants, Immunologic; D000998:Antiviral Agents; D004187:Disaccharides; D004731:Endotoxins; D006017:Glycolipids; D007375:Interleukin-1; D011749:Pyrogens; D013403:Sugar Phosphates; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D005688:Galactosamine; C044283:lipid X; D015232:Dinoprostone