3-O-Desacyl monophosphoryl lipid A derivatives: synthesis and immunostimulant activities.

The synthesis of a series of novel analogues of lipid A, the active principle of lipopolysaccharide, is reported. In these compounds, the 1-O-phosphono and (R)-3-hydroxytetradecanoyl moieties of native Salmonella minnesota R595 lipid A have been replaced with hydrogen and the length of the normal fatty acyl residues has been systematically varied. Normal fatty acid chain length in the 3-O-desacyl monophosphoryl lipid A (MLA) series is shown to be a critical determinant of iNOS gene expression in activated mouse macrophages and the induction of proinflammatory cytokines in human peripheral monocytes. Examination of pyrogenicity in rabbits and lethal toxicity in D-galactosamine-treated mice shows that toxic effects in the MLA series can be ameliorated by modifying fatty acid chain length. When used as an adjuvant for tetanus toxoid vaccines, certain MLA derivatives enhance the production of tetanus toxoid-specific antibodies in mice.

Monophosphoryl lipid A as a prophylactic for sepsis and septic shock.

The ability of monophosphoryl lipid A (MLA) to provide prophylactic protection against septic shock was evaluated in a mouse model of induced endotoxin hypersensitivity. Treatments of hypersensitized animals with low doses of MLA attenuated endotoxin lethality and endotoxin-mediated liver damage. These effects were related to the ability of MLA to suppress accumulation of TNF-alpha and IFN-gamma in the bloodstream of animals. MLA treatments had only a modest effect in suppressing the accumulation of nitrate in the bloodstream. This implied that MLA did not suppress induction of macrophage and hepatocyte nitric oxide synthetases that contribute to antimicrobial defense and protect against endotoxin-mediated liver damage. The MLA treatments did not appear to compromise inflammatory defenses against local infection since locally recruited leukocytes remained responsive to endotoxin after hypersensitivity had been attenuated. In agreement with these findings, other studies have shown that the induction of endotoxin tolerance by MLA parallels the induction of resistance of animals to lethal challenges with either Gram negative or Gram positive bacteria. As predicted from preclinical studies, human trials of the clinical form of MLA (MPL-immunostimulant) have confirmed that MLA could attenuate systemic responses to endotoxin in normal volunteers, including the attenuation of blood cytokine accumulation and attenuation of symptomatic responses.

Activation of arachidonic acid metabolism in mouse macrophages by bacterial amphiphiles.

The relative activities of lipoteichoic acid (LTA) from four Gram-positive bacteria were compared to different lipopolysaccharide (LPS) preparations for activation of arachidonic acid metabolism in mouse peritoneal macrophages. Total eicosanoid was determined in cultures labeled with [3H]-arachidonic acid. Prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) were determined by EIA analysis. The relative potencies of the different preparations were: smooth LPS from Salmonella abortus > or = Re-LPS from Salmonella minnesota (R-595) > or = LTA from Streptococcus pyogenes approximately Streptococcus faecalis approximately Staphylococcus aureus > or = monophosphoryl lipid A derived from the Re-LPS >> LTA from Bacillus subtilis. Activation of eicosanoid release was inhibited by staurosporin for all of the amphiphiles tested. Treatment of the macrophage cultures with LTA from S. pyogenes, S. faecalis, and S. aureus, either in the presence or absence of indomethacin, desensitized the cells to eicosanoid release on subsequent challenge with LPS. The desensitized cells remained responsive to the phorbol ester phorbol myristate acetate. LPS from Gram-negative bacteria has immunostimulatory and endotoxic activities which result, in part, from the release of eicosanoids and other mediators from activated macrophages. The similarities in the patterns of cell activation by LPS and LTA suggest that lipoteichoic acids might contribute to the pathogenicities of Gram-positive bacteria.

Effects of tumor necrosis factor and dexamethasone on the regulation of interferon-gamma induction by monophosphoryl lipid A.

Monophosphoryl lipid A (MLA), derived from lipopolysaccharide (LPS) of Salmonella minnesota strain R595, induced rapid accumulation of interferon (IFN)-gamma in mice. Tumor necrosis factor (TNF)-alpha appeared to be a cofactor for IFN-gamma induction by MLA. With low doses of MLA (< 5 micrograms), IFN-gamma induction was dependent upon exogenous TNF-alpha administered either in advance of or with MLA. A 25 micrograms dose of MLA induced significant IFN-gamma accumulation in the absence of exogenous TNF-alpha. In this case, endogenous TNF-alpha appeared to be a cofactor in the response, since suppression of TNF-alpha production with dexamethasone inhibited IFN-gamma induction, and this inhibition was overcome by administration of exogenous TNF-alpha with MLA. Treatment of animals with MLA tolerized them against LPS. Tolerant animals did not produce IFN-gamma when challenged with LPS, and this tolerance was not abrogated by supplementing mice with exogenous TNF-alpha during the challenge. Although dexamethasone inhibited IFN-gamma induction by MLA, it did not inhibit tolerance induction by MLA.

A rationale for the prophylactic use of monophosphoryl lipid A in sepsis and septic shock.

Monophosphoryl lipid A (MLA), a substructure of bacterial lipopolysaccharide (LPS), is being developed as a prophylactic for sepsis and septic shock. In the present study it was shown that MLA induced a rapid accumulation of IFN-gamma in mice that correlated with an in vivo priming of macrophages. Primed macrophages could be induced in vitro to synthesize nitric oxide, a key mediator of macrophage cytotoxicity. Due to its rapid clearance, MLA was not present in circulation at the time when IFN-gamma accumulated, suggesting that MLA could not synergize with IFN-gamma to systemically activate macrophages in vivo. MLA treatment tolerized mice against the IFN-gamma response--ie., treatment of mice with MLA on day 1 blocked LPS from inducing IFN-gamma on days 2-4. The significance of these results in relation to MLA's ability to enhance non-specific resistance and block LPS lethality in animals is discussed.

Dissociation of proteinase-inhibitor complexes by trichloroacetate.

It was demonstrated that the addition of high concentrations of the chaotrope, sodium trichloroacetate, to proteinase assays provided for a dissociation of proteinase-inhibitor complexes. The complexes evaluated contained a heat-stable, polypeptide inhibitor of cysteine proteinases isolated from the cellular slime mold, Dictyostelium discoideum. The proteinases that were present in separate complexes included either D. discoideum proteinases or the plant proteinase papain. The general assay procedures described may be useful in detection of endogenous proteinase-inhibitor complexes in many systems.

Use of a Western blotting technique in the purification of a cysteine proteinase inhibitor.

In Western blotting procedures, proteins are resolved in sodium dodecyl sulfate-polyacrylamide gels with subsequent electrophoretic transfer onto nitrocellulose membranes. Although this procedure is generally employed as an analytical technique for assessing interactions of proteins with antibodies, the present report describes the use of Western blotting as a preparative procedure in the purification of a biologically active proteinase inhibitor from the cellular slime mold, Dictyostelium discoideum. The feasibility of using Western blotting for inhibitor purification depended upon the unique stability properties of the inhibitor under denaturing conditions.

Antibodies that recognize phosphodiester-linked amino-N-acetylglucosamine-1-phosphate residues.

An affinity chromatography procedure was developed for isolating antibodies that recognized phosphodiester-linked alpha-N-acetylglucosamine-1-phosphate (alpha-GlcNAc-1-P) residues. The affinity resin consisted of uridine-5'-diphospho-alpha-N-acetylglucosamine (UDPGlcNAc) conjugated to Sepharose. Antiserum prepared against Proteinase 1 from the cellular slime mold, Dictyostelium discoideum was used as a source of the anti-alpha-GlcNAc-1-P antibodies. Immunoblot assays showed that the affinity-isolated antibodies recognized phosphoglycosylated subunits of Proteinase 1, and that UDPGlcNAc blocked this interaction.

Occurrence of N-acetylglucosamine-1-phosphate in proteinase I from Dictyostelium discoideum.

A previous study (Gustafson, G.L., and Thon, L. A. (1979) Biochem. Biophys. Res. Commun. 86, 667-673) demonstrated that Proteinase I from the cellular slime mold, Dictyostelium discoideum, was conjugated with phosphoryl moieties. This report describes a characterization of the covalent structure of these moieties. Essentially all of the phosphate associated with purified enzyme was released as a sugar-phosphate during mild alkaline hydrolysis. The sugar-phosphate was isolated from alkaline hydrolysates of Proteinase I by Sephadex G-25 chromatography and identified as the alpha-anomer of N-acetylglucosamine-1-phosphate. In separate experiments, involving acid hydrolysis of Proteinase I, it was shown that enzyme-phosphate could also be isolated as O-phosphorylserine. Based on the recovery of O-phosphorylserine from acid hydrolysates, it was concluded that the majority of the N-acetylglucosamine-1-phosphate residues in the proteinase were esterified to peptidyl serines through phosphoester bonds.