Mutations of the gene Lps selectively impede lipopolysaccharide (LPS) signal transduction in C3H/HeJ and C57BL/10ScCr mice, rendering them resistant to endotoxin yet highly susceptible to Gram-negative infection. The codominant Lpsd allele of C3H/HeJ mice was shown to correspond to a missense mutation in the third exon of the Toll-like receptor-4 gene (Tlr4), predicted to replace proline with histidine at position 712 of the polypeptide chain. C57BL/10ScCr mice are homozygous for a null mutation of Tlr4. Thus, the mammalian Tlr4 protein has been adapted primarily to subserve the recognition of LPS and presumably transduces the LPS signal across the plasma membrane. Destructive mutations of Tlr4 predispose to the development of Gram-negative sepsis, leaving most aspects of immune function intact.

A method is described which is specific for the extraction of lipopolysaccharides from R form bacteria. The extraction mixture which is monophasic, consists of aqueous phenol, chloroform and petroleum ether. R form lipopolysaccharides (glycolipids), due to their lipophilic nature, arc completely soluble in the mixture. S and T form lipopolysaccharides as well as proteins, nucleic acids, and polysaccharides, are insoluble, and they are excluded from the extracts. The method is mild, as it can be carricd out at below 10". The yields are generally higher than those obtained by phenol-water extraction, and the products are usually water-soluble. Lipopolysaccharides havc been successfully extracted from all R form bacteria so far attempted.Many methods have been developed for the extraction of endotoxic lipopolysaccharides from Salnzonetla and other gram-negative bacteria [ 1,2]. One of the methods, which is wideIy used, is the phenolwater procedure [3,4], because it is applicable to many different groups of bacteria and because it is one of the few methods by which also lipopolysaccharides from R mutant bacteria can be extracted.I n this method, dried bacteria are treated a t 69" in a mixture of phenol and water (45:55, v/v). On cooling, the mixture, which is monophasic above 68", separates into a phenol phase containing mainly proteins, and a water phase containing lipopolysaccharide and nucleic acid.However, there are recent reports in the literature describing unexpected results. Ashwell et al. , who investigated a Salmmella R mutant, isolated the corresponding heptose-less lipopolysaccharide (glycolipid) from the phenol interphase, though, in our hands, the phenol-water extraction of the same mutant yielded an identical lipopolysaccharide (glycolipid) in the water phase [8], however, in a rather low yield.A11 the Iipopolysaccharides found to be soluble in the phenol phase seem to have the common characteristic of being hydrophobic in nature. The lipopolysaccharides from X . campstris and Citrobacter contain 3-N-acetylamino-3,6-dideoxyhexose [5,9,10] and the hcptose-less lipopolysaccharide of the R mutant contains besides 2-keto-3-deoxyoctonate (KDO) about 700/, of lipid A [8].Unwuul Abbreviation. KDO, 2-keto-3-deoxyoctonate.Europenn J. Biochem., Vol. 9 J 7The present paper describes an extraction method which is specific for R form lipopolysaccharides which are obtained as water-soluble preparations of high purity regarding contamination with proteins and nucleic acid. S form lipopolysaccharides are excluded from the extract, and also certain polymers, like glucans, are not co-extracted. MATERIALS BND METHODS BacteriaThe following S and R strains were used: S. minnesota S form, mR345, mRz, mR595 [ 8 ] , S . godesberg gR15 (isolated by J. Schlosshardt), and S . typhimurium TV 161 1111. They were cultivated as described previously [ 121. After harvesting, the bacteria were washed with distilled water. Saline or any other salt solution should be strictly avoided, as such a treatment may result in a very poor lip...

Treatment of rabbits, rats, and mice with Dgalactosamine increased their sensitivity to the lethal effects of lipopolysaccharide several thousand fold. The susceptibility of the animals was highest when the lipopolysacharide was injected together with galactosamine and decreased successively when injection was carried out 1, 2, and 3 hr later. Sensitization was absent when the lipopolysaccharide was administered 1 hr before or 4 hr after galactosamine. The onset of lethality after treatment with galactosamine and lipopolysaccharide occurred faster than with lipopolysaccharide alone; usually all animals died 5-9 hr later. The galactosamine-induced sensitization to lipopolysaccharide could be reversed by uridine which is known to inhibit the early biochemical alterations induced by the amino sugar in the hepatocytes. Although galactosamine is known to exhibit hepatotoxic activity inducing ultimate necrosis of the hepatocytes, the data so far suggests that the sensitization to lipopolysaccharide is related only to the early metabolic effects ofthe hexosamine.

The recessive mutation 'Heedless' (hdl) was detected in third-generation N-ethyl-N-nitrosourea-mutated mice that showed defective responses to microbial inducers. Macrophages from Heedless homozygotes signaled by the MyD88-dependent pathway in response to rough lipopolysaccharide (LPS) and lipid A, but not in response to smooth LPS. In addition, the Heedless mutation prevented TRAM-TRIF-dependent signaling in response to all LPS chemotypes. Heedless also abolished macrophage responses to vesicular stomatitis virus and substantially inhibited responses to specific ligands for the Toll-like receptor 2 (TLR2)-TLR6 heterodimer. The Heedless phenotype was positionally ascribed to a premature stop codon in Cd14. Our data suggest that the TLR4-MD-2 complex distinguishes LPS chemotypes, but CD14 nullifies this distinction. Thus, the TLR4-MD-2 complex receptor can function in two separate modes: one in which full signaling occurs and one limited to MyD88-dependent signaling.

Allergies to nickel (Ni(2+)) are the most frequent cause of contact hypersensitivity (CHS) in industrialized countries. The efficient development of CHS requires both a T lymphocyte-specific signal and a proinflammatory signal. Here we show that Ni(2+) triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4). Ni(2+)-induced TLR4 activation was species-specific, as mouse TLR4 could not generate this response. Studies with mutant TLR4 proteins revealed that the non-conserved histidines 456 and 458 of human TLR4 are required for activation by Ni(2+) but not by the natural ligand lipopolysaccharide. Accordingly, transgenic expression of human TLR4 in TLR4-deficient mice allowed efficient sensitization to Ni(2+) and elicitation of CHS. Our data implicate site-specific human TLR4 inhibition as a potential strategy for therapeutic intervention in CHS that would not affect vital immune responses.

Endotoxins (lipopolysaccharides, LPS) are the main factors of pathogenicity of Gram-negative bacteria . In experimental animals, they induce a broad spectrum of pathophysiological reactions, many of them being similar to those manifested during infection, and which may lead to shock and ultimately death.Different mammalian species show large differences in their susceptibility to the lethal effects of endotoxin. The natural sensitivity to endotoxin may be increased in a variety of experimental models . One of these, treatment with Dgalactosamine, increases the sensitivity of mice to the lethal effects of endotoxin more than 100,000-fold (I). D-Galactosamine is a hepatotoxic agent, its effects being confined to hepatocytes. The early biochemical effects on hepatocytes, which are necessary for the development of sensitization to endotoxin, are depletion of UTP and changes in uracyl nucleotides that result in an impaired biosynthesis of macromolecular cell constituents (RNA, membrane glycoproteins, glycogen, etc.) (2).The above metabolic changes in hepatocytes also occur in endotoxin-resistant C3H/HeJ mice after treatment with D-galactosamine; however, sensitization to endotoxin is not demonstrable due to the absence of endotoxin sensitive macrophages . Transfer of a relatively small number (2 X 107) of macrophages obtained in culture from bone marrow precursor cells of endotoxin-sensitive C3H/HeN mice rendered D-galactosamine-treated C3H/HeJ mice sensitive to the lethal effect of as little as I ug LPS (3). This provided direct evidence for the central role of macrophages in mediating endotoxin reactions. It further demonstrated that D-galactosamine sensitization was not due to an enhancement of the actual mechanisms of endotoxicity (e.g., hyperreactivity of macrophages), but to the lowering of the threshold of susceptibility to the toxic products of macrophages. Although the actual mechanisms of endotoxicity are not known, there exists general agreement today that toxicity is caused by endogenous mediators, which are released on interaction of endotoxin with target cells.In the past, a number of mediators mainly of macrophage origin have been discussed in relation to endotoxic reactions. Recently, Beutler et al. (4) have provided evidence that cachectin, which is identical with tumor necrosis factor

The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity. It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests.Lipopolysaccharides (endotoxins) of gram-negative bacteria which consist of a heteropolysaccharide and a lipid component (termed lipid A) elicit multiple acute pathophysiological effects such as fever, lethality, Shwartzman reactivity, macrophage and B-lymphocyte activation, and other activities [I]. In 1954 it was proposed that for the induction of these effects the polysaccharide portion is dispensable and that the lipid A component represents the active center responsible for the endotoxic properties of lipopolysaccharides [2]. Evidence for this was then obtained in numerous investigations [2 -41 and this concept is now generally accepted.The chemical structure of the lipid A component of several enterobacterial lipopolysaccharides has been analysed during recent years in great detail (for reviews see [5, 61) and it was recognized that lipid A of Escherichiu coli possesses a comparatively simple structure. Free E. coli lipid A consists of a 8(1-6)-linked D-glucosamine disaccharide which is substituted by two phosphoryl groups, one being bound to position 4' of the nonreducing glucosamine residue (GlcN 11) and one being a-linked [7] to the glycosidic hydroxyl group of the reducing glucosaminyl group (GlcN I) (Fig.

The essential role of Toll-like receptors (TLR) in innate immune responses to bacterial pathogens is increasingly recognized, but very little is known about the role of TLRs in host defense against infections with eukaryotic pathogens. For the present study, we investigated whether TLRs contribute to the innate and acquired immune response to infection with the intracellular protozoan parasite Leishmania major. Our results show that TLR4 contributes to the control of parasite growth in both phases of the immune response. We also addressed the mechanism that results in killing or growth of the intracellular parasites. Control of parasite replication correlates with the early induction of inducible nitric oxide synthase in TLR4-competent mice, whereas increased parasite survival in host cells from TLR4-deficient mice correlates with a higher activity of arginase, an enzyme known to promote parasite growth. This is the first study showing that TLR4 contributes to the effective control of Leishmania infection in vivo.