Selective refractoriness of macrophages to endotoxin-induced production of tumor necrosis factor, elicited by an autocrine mechanism.

Tolerance of macrophages to endotoxin (lipopolysaccharide, LPS) can be induced in vitro by LPS itself. We show here that one of the mechanisms of tolerance to LPS can be mediated via an autocrine process. Continuous exposure to LPS is not required to induce macrophage desensitization. Refractoriness to production of tumor necrosis factor (TNF) in response to LPS can be transferred from tolerant to naive macrophage populations by incubation of the latter with the culture supernatant of the former, in the absence of endotoxin. The active factor present in this macrophage-desensitizing culture supernatant (MD-Sup) is more efficiently removed by incubation with tolerant macrophages than by incubation with naive macrophages. The refractoriness elicited by treatment with MD-Sup is restricted to a decreased TNF response to LPS; interleukin-1 (IL-1) and IL-6 responses are not affected.

Specific binding of lipopolysaccharides to mouse macrophages--I. Characteristics of the interaction and inefficiency of the polysaccharide region.

Tritium-labeled lipopolysaccharide interacted specifically and reversibly with mouse peritoneal macrophages. The binding was higher at 22 degrees C than at 4 degrees C, but was no longer observable at 37 degrees C. The specificity of the interaction (inhibition with unlabeled LPS) was strictly dependent on the presence of serum, and required divalent cations. The binding was saturable. The specific binding sites of peritoneal macrophages were saturated with 6-9 x 10(6) LPS molecules/cell, and those of macrophage-like cell lines with 2-3 x 10(6) molecules/cell. The binding of LPS was not inhibited by ligands of scavenger receptors (maleylated BSA) or complement receptors (zymosan), but was strongly inhibited with dexamethasone, a glucocorticoid which is known to modulate the expression of other surface markers of macrophages. The polysaccharide region of the LPS, as well as 3-deoxy-2-octulosonic acid (KDO) coupled to bovine serum albumin, did not bind to macrophages, whereas a specific binding was observed with a lipid A-BSA conjugate.

Specific binding of lipopolysaccharides to mouse macrophages--II. Involvement of distinct lipid a substructures.

The interaction of lipopolysaccharide-binding sites of mouse macrophages with the Lipid A region of endotoxins (LPS) was demonstrated by direct binding of labeled Lipid A conjugates, by inhibition of the binding of labeled LPS with anti-Lipid A monoclonal antibodies, and by the considerable reduction of this binding after chemical and enzymatic removal of the fatty acid esters of the LPS. The substructures of Lipid A required for the specific binding of LPS to macrophages were analyzed by the use of synthetic lipids consisting of mono- or disaccharide derivatives of glucosamine. The two phosphate groups of Lipid A (at positions 1 and 4') as well as certain hydroxyl groups, appeared to play a critical role in the binding. However, the reactivities of the synthetic lipids with the macrophage surface, as compared with those with anti-Lipid A antibodies, could hardly be explained by the existence of a single LPS receptor, and suggested the presence, on the macrophage surface, of different LPS-binding sites that recognize different substructures or spatial configurations of the lipid moiety of endotoxins.

Chemical and serological characterization of the Bordetella hinzii lipopolysaccharides.

Bordetella hinzii has recently been isolated from immunocompromised human hosts. The polysaccharides isolated from its endotoxin (lipopolysaccharide, LPS) were investigated using chemical analyses, NMR, gas-liquid chromatography/mass spectrometry and mass spectrometry by plasma desorption, matrix-assisted laser desorption/ionization and electrospray. The following structure for the O-chain-free LPS was deduced from the experimental results: carbohydrate structure [see text] Mass spectrometry and serology revealed that the O-chains were different from the homopolymer common to Bordetella bronchiseptica and Bordetella parapertussis strains and were composed of a trisaccharide repeating unit. Masses up to 8 kDa were obtained for native LPS molecular species.

Preparative scale separation of functionally distinct murine B cell subpopulations.

Mouse splenic B cells were found to be rapidly and reproducibly fractionated by adherence at 4 degrees C to plastic dishes pretreated with an alkaline buffer. The cell subsets obtained exhibited marked differences in size, surface immunoglobulins (sIg), and responses to B mitogens. Adherent B lymphocytes, bearing high densities of sIg, responded particularly to LPS whereas non-adherent B lymphocytes, with low densities of sIg, responded especially to dextran sulfate. The correlations between the separated populations and subsets at distinct maturation stages are discussed.

Down-modulation through protein kinase C-alpha of lipopolysaccharide-induced expression of membrane CD14 in mouse bone marrow granulocytes.

We have previously shown that stimulation of mouse bone marrow granulocytes (BMC) by lipopolysaccharide (LPS) induces the expression of CD14. We found here that phorbol 12-myristate 13-acetate (PMA) blocks this LPS effect. The aim of this study was to investigate the mechanism by which PMA can block the LPS signaling pathway in BMC. The unmodified binding of a radiolabeled LPS in PMA-treated cells indicated that the PMA effect was not the consequence of a shedding or an internalization of the LPS receptor, but was rather due to a biochemical event that follows the interaction of LPS with its receptor. The observations that a selective activator of protein kinase C (PKC)-alpha (sapintoxin D) mimics the PMA effect, whereas a selective PKC-alpha inhibitor (Ro-320432) antagonizes this effect, suggest a regulatory role of PKC-alpha in the LPS signaling pathway in mouse BMC.

The splenic pool of mouse stem cells: in vitro differentiation and expression of the BP-1 alloantigen on cells of the lymphoid and myeloid lineages.

The relative paucity of data about the development of the stem cell pool present in the spleen prompted this study. During in vitro cultures of B-enriched lymphocytes from mouse spleens and in the presence of a culture supernatant of WEHI-3 cells (WEHI-SUP), a population of cells expressing the BP-1 antigen appears progressively, reaches an optimal size 8 days after initiation of the culture, and disappears on day 28. In 8-day-old cultures, a minor population of cells bearing both BP-1 and B220 can be detected. The growth of this cell population, with characteristics of the B lymphoid lineage (pro-B), is strictly dependent on the presence of WEHI-SUP in the medium. After 2 weeks of culture, the BP-1 antigen is expressed on a cell population, which is essentially constituted of B220-, polynuclear cells. The BP-1 antigen, which is considered as characteristic of early cells of the B lymphoid lineage, can therefore also be expressed on cells of the myeloid lineage. The injection of BP-1+ or B220+ cells in irradiated mice can hardly reconstitute their B cell pool, whereas BP-1- and B220- cells are much more efficient in vivo progenitors of this cell lineage.

Variation in Bordetella bronchiseptica lipopolysaccharide during human infection.

We previously reported the case of a human chronic Bordetella bronchiseptica respiratory infection, due to contact with infected rabbits. Lipopolysaccharides of the human isolates, of one rabbit isolate and of isolate from other origins were analyzed with sera from infected mice, rabbit and human. Antigenicity and length of the lipopolysaccharide molecules varied between isolates. We showed a progressive loss of O-chain during infection, associated with an enhanced susceptibility of the isolates to the bactericidal effect of normal serum. This observation suggests the existence of an intracellular niche which selects for strains with distinct lipopolysaccharide types.

Characterisation of ovine Bordetella parapertussis isolates by analysis of specific endotoxin (lipopolysaccharide) epitopes, filamentous haemagglutinin production, cellular fatty acid composition and antibiotic sensitivity.

Isolates of Bordetella parapertussis, recovered from sheep or man, were characterised by reaction with specific anti-Bordetella lipopolysaccharide monoclonal antibodies, production of filamentous haemagglutinin, fatty acid patterns, and antibiotic sensitivity. Generally, the isolates lay within one of four groups, with separation of the ovine isolates into two groups. Reactions with specific monoclonal antibodies against lipopolysaccharide separated the ovine isolates into these two groupings. Analysis of the cellular fatty acid compositions by cluster analysis differentiated between the human and the ovine strains and also showed variation within the ovine isolates. When the production of filamentous haemagglutinin was analysed in an ELISA system, a similar pattern emerged. Varying concentrations of filamentous haemagglutinin (11-429 ng (mg total protein)-1) were extracted from the human isolates and the one group of ovine isolates with no significant protein detected in the other ovine group. These studies demonstrate variation between and within B. parapertussis isolates recovered from two mammalian sources.

Protein phosphorylation pathways involved during lipopolysaccharide-induced expression of CD14 in mouse bone marrow granulocytes.

Lipopolysaccharide (LPS) of Gram-negative bacteria interacts with a CD14-independent receptor of mouse bone marrow granulocytes (BMC), and triggers in these cells the expression of CD14, an inducible type of LPS receptor (iLpsR). This particular response of BMC to LPS required the activation of protein tyrosine kinase and p38 MAP kinase. The inhibition of the LPS effect by the MEK inhibitor PD-98059 suggested that the ERK pathway was also involved. Unexpectedly, protein kinase C, myosin light chain kinase, cAMP-, cGMP-, and Ca(2+)/calmodulin-dependent kinases, as well as ecto-protein kinases, were not required for iLpsR expression. However, other yet unidentified serine/threonine protein kinase(s) were implied since the BMC response to LPS was markedly reduced after exposure to three inhibitors of such kinases (K-252a, H-7, and KT-5823). The atypical kinase requirements observed in this study may be due either to a novel signaling LPS receptor complex present in BMC, or to the particular events involved in CD14 biosynthesis.

Structural features involved in the mitogenic activity of Bordetella pertussis lipopolysaccharides for spleen cells of C3H/HeJ mice.

Spleen cells from the C3H/HeJ mouse strain cannot be stimulated by many smooth-type lipopolysaccharides (LPSs), and by the main biologically-active region (lipid A) of these molecules. The genetic origin of this defect (expression of the mutant allele Lpsd at the chromosome 4 locus) was established over 20 years ago, but its biochemical nature has remained undefined. Several investigators have noted, however, that some particular LPSs can bypass this defect, and stimulate the proliferation of C3H/HeJ B lymphocytes. In this study we compare the mitogenic activities of the LPSs isolated from a wild strain (1414) and from a mutant 'rough' strain (A100) of Bordetella pertussis. Both LPS-1414 and LPS-A100 were mitogenic for C3H/HeJ spleen cells, but their lipid A fragments were not. This indicates that a carbohydrate structure proximal to lipid A is involved in the mitogenic activity. However, the isolated polysaccharides were not mitogenic. Four sugars are common to both LPS-1414 and LPS-A100: an heptose, and three sugars bearing free amino groups. After removal of these four sugars from the LPSs by nitrous acid treatment, the recovered lipooligosaccharides were not mitogenic in Lpsd spleen cells. The results suggest that substructures present in lipid A and in this group of four sugars are both required for induction of a mitogenic effect in Lpsd splenocytes, whereas lipid A alone can stimulate Lpsn spleen cells.

Effect of synthetic lipids on apoptosis and expression of alkaline phosphatase in B-lymphocytes: influence on lipopolysaccharide action.

Synthetic lipids were examined for their ability to mimic or to antagonize lipopolysaccharide (LPS) action in murine B-lymphocytes. Several recognized effects of LPS were analyzed: prevention of spontaneous apoptosis, expression of alkaline phosphatase (ALP) and stimulation of proliferation. Three synthetic lipids were used for that purpose: a lipopeptide (compound MTPP) which carries non-hydroxylated fatty acids, and is thus unrelated to LPS, and two glycolipids with hydroxylated fatty acids (compounds D2 and PPDm2-B), structurally related to the lipid A region of enterobacterial and Rhodopseudomonas LPS, respectively. We found that the ability of these lipids to induce LPS-like responses was not correlated with their structural analogy with LPS. Thus, the lipopeptide, MTPP, mimicked LPS in the three activities, whereas the glycolipid, D2, did not. In contrast, the ability of synthetic lipids to block LPS effects was correlated with their structural analogy with LPS. We thus observed that compound D2 selectively blocked LPS-induced ALP expression and that PPDm2-B selectively inhibited LPS-induced prevention of apoptosis. These synthetic lipids could therefore be useful for studying the LPS-mediated signals involved in B-cell activation and apoptosis.

Interaction of lipopolysaccharides with cells of immunological interest.

Recent progress has been made in understanding the diverse effects of bacterial endotoxin (LPS) on animal systems. Experiments from our laboratory, as well as those from several other groups, have provided strong evidence supporting the presence of different sites of interaction with LPS in cells of immunological interest. However, the sequence of expression of receptors, and their interrelations, are poorly understood. In this review, we summarize the essentials of these studies, with particular emphasis on experiments from our own laboratory. Our results suggest that the "positive" signals which trigger the activation of B lymphocytes and bone-marrow cells, as well as the "negative" signals which trigger macrophage desensitization and tolerance to LPS, might be delivered by sparsely represented and yet unidentified LPS receptors. The identification of these molecules would clarify the mechanisms involved in the immunostimulatory as well as in the pathophysiological effects of endotoxin, and should have significant impact upon potential therapeutic intervention in endotoxin-mediated disease.

Preparation and binding specificity of a monoclonal antibody recognizing 3-deoxy-D-manno-2-octulosonic acid (Kdo) in lipopolysaccharides of Re chemotype.

A mouse monoclonal antibody (MAb E1) was raised against the lipopolysaccharide (LPS) of the Re mutant R595 of Salmonella minnesota. This IgG3 antibody (MAb E1), unstable at low pH and low ionic strength, was purified by chromatography on QAE Sepharose A50. The binding specificity of MAb E1 was characterized by direct and inhibition enzyme immunoassays, using natural LPSs from different strains and chemotypes, and synthetic analogs of LPS substructure of the 3-deoxy-D-manno-2-octulosonic acid (Kdo) and Lipid A regions. Among various LPSs, MAb E1 reacted exclusively with those of Re-chemotype. It recognized alpha-Kdo- monosaccharide and disaccharide structures present as non-reducing side chains in various Re-type LPSs and synthetic antigens. The antibody did not react with Lipid A or various lipids, and the presence of the lipid region was not necessary for the reaction. The recognition of the epitope was not reduced by the presence of a substituent at O-8 of one of the two Kdo units present in the Re LPS from Proteus mirabilis, but the reaction was inhibited by phosphorylation of O-4 of Kdo, by the proximity of core (heptose) or Lipid A (acylated glucosamine) residues, or by certain LPS-LPS interactions.

Lipopolysaccharide-binding molecules: transporters, blockers and sensors.

Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, can be beneficial to the host by activating the innate immune system, or harmful, by inducing inflammation, disseminated intravascular coagulation, multiple organ failure, shock and often death. On the bacteria, and in host biological fluids and cells, LPS is never free but constantly attached to cognate-binding proteins. Understanding how LPS is transported and further recognized by sensors able to deliver a signal, or by inactivating molecules able to neutralize its biological effects, is an important goal. This review describes the large panel of peptides and proteins reported to associate with LPS, and provides information on their origin, their structure and the location of amino acid residues involved in their interaction with LPS. A better understanding of the mode of recognition of LPS by cognate proteins prompted many laboratories to design on a rational basis synthetic molecules which can be used to detect low amounts of endotoxin, or to act as efficient blockers of in vitro and in vivo responses to LPS.

Strategies for the control of LPS-mediated pathophysiological disorders.

Lipopolysaccharides released from Gram-negative bacteria after infection initiate an alarm response in the host, which has supposedly evolved to protect it. However, an exaggerated response leads to a cascade of pathophysiological events termed sepsis. In the USA alone, the annual number of deaths caused by sepsis ( approximately 70,000) is comparable with that caused by AIDS. The author describes the major advances of knowledge in this field and the attempts to convert this into successful therapeutics. Anti-endotoxin and anti-inflammatory agents have been disappointing, but new strategies might result in effective treatments in the forthcoming years.

Endotoxin-induced tumor necrosis factor (TNF): selective triggering of TNF and interleukin-1 production by distinct glucosamine-derived lipids.

The isolated lipid A of Bordetella pertussis endotoxin (LipA) has been found to induce in vitro release of tumor necrosis factor (TNF) by murine macrophages, albeit much less efficiently than does the intact lipopolysaccharide. Synthetic analogs (monosaccharides M4 and M6) of both glucosamine units present in the LipA backbone induced production of TNF by peritoneal macrophages of Swiss mice. Macrophages from A/J mice gave higher responses than those from Swiss mice, while those of C3H/HeJ mice were unresponsive. Enhancement of TNF secretion was observed for all cells if they were pretreated with a calcium ionophore, and no otherwise inactive substance became active with cells thus treated. For synthetic monosaccharide derivatives, a phosphate group on O-4 was not required for, and a phosphate group on O-1 abolished, the TNF-inducing activity. Synthetic monosaccharides, chemically closely related to substructures recognized to be present in isolated lipid A preparations, could induce either TNF or interleukin-1 (IL-1) production, but not both simultaneously: the monosaccharides M4 and M6 were active TNF inducers, but did not initiate IL-1 production, while the monosaccharides M9 and lipid X efficiently elicited IL-1 production, but did not trigger TNF secretion. It should be noted, however, that the active synthetic compounds are considerably less efficient TNF inducers as is the intact B. pertussis endotoxin.

Inhibition of lipopolysaccharide mitogenicity with characterized anti-lipid A monoclonal antibodies.

Antibodies to lipid A were raised in mice immunized with non-hydrolysed Bordetella pertussis microorganisms, coated with lipid A isolated from the same bacteria. Anti-lipid A activity of immune sera was measured by radioimmunoassay. Four hybrid cell lines that secrete antibodies directed against the hydrophobic region of B. pertussis lipopolysaccharide were produced by cell fusion between myeloma cells and spleen cells from immunized C3H/He-PAS mice. Differences were observed in the potency of the isolated monoclonal antibodies to inhibit B cell proliferation induced by lipopolysaccharide (LPS) or by lipid A, suggesting a selective recognition of effector sites present on the hydrophobic region of LPS.