Endotoxin: physical requirements for cell activation.

Lipopolysaccharide (LPS) is the eminent lipid component of the outer leaflet of the outer membrane of Gram-negative bacteria and the major initiator of innate immune response to bacterial infection. Below the critical micellar concentration (CMC), LPS is exclusively present as a monomer. Above this concentration, aggregates are formed. Increasing the concentration beyond the CMC leads to an increase in aggregate concentration, whereas the concentration of monomers remains constant or even decreases. The question how LPS activates immune cells and whether the aggregate or the monomer is the biologically active unit has been and still is controversial. To prepare clearly defined monomeric solutions, we utilized a dialysis set-up consisting of a donor and an acceptor chamber, separated by a dialysis diaphragm with a cut-off of 5 kDa, thus allowing only monomers to pass. Human mononuclear cells (MNCs) were then stimulated with equal concentrations of aggregates and monomers, respectively, of deep rough mutant LPS from Escherichia coli strain F515 (Re LPS) and TNF-alpha release was determined. In contrast to earlier and very recent work of others, we started with a preparation of aggregate-suspensions and pure monomer-solutions and show that monomers are significantly less active than aggregates in the absence and presence of serum proteins at identical concentrations. In our model, we propose that LPS aggregates are detected by membrane-associated LBP and intercalated into the cell membrane to bring LPS into close proximity to signaling proteins in the membrane, thus finally leading to cell activation. To support this model, we present data showing that LBP is indeed present in or at the cell membrane of human macrophages.

Structural polymorphism and endotoxic activity of synthetic phospholipid-like amphiphiles.

The physicochemical characteristics and in vitro biological activity of various synthetic hexaacyl phospholipid dimers were compared with the respective behavior of bacterial endotoxins (lipopolysaccharide, LPS). The structural variations of the synthetic amphiphiles include different stereochemical (R,S) configurations about their ester- and amide-linkages for the acyl chains and differences in the length of the serine backbone spacer. The temperature of the gel to liquid crystalline phase transition of the acyl chains (T(c)) lies between 10 and 15 degrees C for the compounds with the shortest backbone and decreases rapidly for the compounds with longer backbones. The phase transition enthalpies (8-16 kJ x mol(-1)) are considerably lower than those of lipid A from hexaacyl endotoxins (28-35 kJ x mol(-1)). In contrast, the dependence of T(c) on Mg(2+) and water content shows a behavior typical for endotoxins: a significant increase with increasing Mg(2+) and decreasing water concentrations. The aggregate structure is sensitively dependent not only on the length of the backbone spacer but also on the different stereochemical variations. It can be directly correlated with the biological activity of the compounds. Thus, as with natural lipid A, the capacity to induce cytokine production in mononuclear cells is directly related to the affinity to form nonlamellar cubic or inverted hexagonal H(II) aggregate structures. Together with the data on the transport and intercalation of the dimers into phospholipid liposomes mediated by the lipopolysaccharide-binding protein (LBP), our conformational concept of endotoxicity and cell activation can be applied to these non-LPS structures: endotoxically active compounds incorporate into membranes of immune cells and cause conformational changes at the site of signaling proteins such as Toll-like receptors or K(+)-channels due to their conical molecular shape.

The role of membrane-bound LBP, endotoxin aggregates, and the MaxiK channel in LPS-induced cell activation.

We have previously shown in patch-clamp experiments on excised outside-out cytoplasmic membrane patches from human macrophages that the activation of a high-conductance Ca(2+)- and voltage-dependent potassium channel, the MaxiK channel, is an early step in LPS-induced transmembrane signal transduction in macrophages. MaxiK can be activated by agonistically active LPS, and activation can be completely inhibited by LPS antagonists (e.g. synthetic compound 406) and by anti-CD14 antibodies. Furthermore, by inhibiting MaxiK with the specific MaxiK blocker paxilline, we could show that activation of MaxiK is essential for LPS-induced cytokine production. As shown by RT-PCR, blockade of MaxiK by paxilline also inhibits induction of the mRNA of TNF-alpha and IL-6. This observation together with the fact that all patch-clamp experiments were done on excised outside-out patches reveal that MaxiK activation is an early step in cell activation by endotoxins. Thus, since cells lacking TLR4 on their surface can also not be activated to produce cytokines, these data allow the conclusion that TLR4 and MaxiK are both essential for activation by LPS and may form a co-operative signaling complex. We have also shown that LBP not only exists as a soluble acute-phase serum protein, but is also incorporated as a transmembrane protein (mLBP) in the cytoplasmic membrane of MNC; in this configuration, it is obviously involved in the binding of endotoxin and its transfer to the transmembrane signaling proteins finally triggering cell activation. Complexation of soluble LBP and LPS in the serum prior to binding of LPS to mLBP, in contrast, leads to neutralization of LPS. Here, we provide evidence from fluorescence resonance energy transfer spectroscopy that endotoxin aggregates are intercalated into reconstituted membranes by mLBP. In addition, cell culture assays and patch-clamp experiments demonstrate that endotoxin activates macrophages and the MaxiK channel in the aggregated, but not in the monomeric, state at similar concentrations.

Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure.

Pathogenic biotypes of Yersinia enterocolitica (serotypes O:3, O:8, O:9, and O:13), but not environmental biotypes (serotypes O:5, O:6, O:7,8, and O:7,8,13,19), increased their permeability to hydrophobic probes when they were grown at pH 5.5 or in EGTA-supplemented (Ca(2+)-restricted) media at 37 degrees C. A similar observation was also made when representative strains of serotypes O:8 and O:5 were tested after brief contact with human monocytes. The increase in permeability was independent of the virulence plasmid. The role of lipopolysaccharide (LPS) in this phenomenon was examined by using Y. enterocolitica serotype O:8. LPS aggregates of bacteria grown in acidic or EGTA-supplemented broth took up more N-phenylnaphthylamine than LPS aggregates of bacteria grown in standard broth and also showed a marked increase in acyl chain fluidity which correlated with permeability, as determined by measurements obtained in the presence of hydrophobic dyes. No significant changes in O-antigen polymerization were observed, but lipid A acylation changed depending on the growth conditions. In standard medium at 37 degrees C, there were hexa-, penta-, and tetraacyl lipid A forms, and the pentaacyl form was dominant. The amount of tetraacyl lipid A increased in EGTA-supplemented and acidic media, and hexaacyl lipid A almost disappeared under the latter conditions. Our results suggest that pathogenic Y. enterocolitica strains modulate lipid A acylation coordinately with expression of virulence proteins, thus reducing LPS packing and increasing outer membrane permeability. The changes in permeability, LPS acyl chain fluidity, and lipid A acylation in pathogenic Y. enterocolitica strains approximate the characteristics in Yersinia pseudotuberculosis and Yersinia pestis and suggest that there is a common outer membrane pattern associated with pathogenicity.

Lack of interaction of fluoroquinolones with lipopolysaccharides.

Fluoroquinolones are known to chelate with di- and trivalent cations, and it has accordingly been claimed that they perturb the integrity of the outer membrane (OM) of gram-negative bacteria. So far, chelation has not been assessed in biologically relevant test systems. Therefore, we investigated the interaction of ciprofloxacin and moxifloxacin in the absence and presence of Mg2+ with whole bacteria and isolated lipopolysaccharide (LPS) from various rough mutant strains of Salmonella enterica chemotypes by applying different biophysical techniques. We found that the fluoroquinolones did not disturb the integrity of the OM and neither were incorporated into LPS monolayers nor displaced Ca2+ from LPS monolayers, suggesting that chelation of fluoroquinolones with divalent cations does not contribute to the antibacterial effect of fluoroquinolones.

A study on the C-terminal membrane anchoring of Escherichia coli penicillin-binding protein 5.

Escherichia coli penicillin-binding protein 5 (PBP5) anchors to the inner membrane in a pH-dependent manner via a C-terminal amphiphilic alpha-helix. Low pH was found to enhance both levels of PBP5 membrane anchoring and levels of alpha-helicity in an aqueous PBP5 C-terminal homologue, which led to the suggestion that levels of PBP5 membrane anchoring are related to levels of PBP5 C-terminal alpha-helicity. Here we have used Fourier-transformed infrared spectroscopy (FTIR) and a peptide homologue of the PBP5 C-terminal sequence to investigate the effect of pH on the conformational behavior of this sequence at a lipid interface and on its ability to interact with lipid. Our results suggest that the membrane-anchoring mechanism of PBP5 is unlikely to involve conformational change in the protein's C-terminal region and may therefore involve conformational changes in the protein's ectomembranous domain.

A K+ channel is involved in LPS signaling.

We previously showed a clear correlation between the molecular conformation of the lipid A moiety of endotoxin molecules and their cytokine-inducing capacity in mononuclear cells. While conically shaped lipid A moieties exhibit a high agonistic activity, a shift to a more cylindrically shaped lipid A leads to a decrease in agonistic and increase in antagonistic activity of the endotoxin molecules. Here, we show the involvement of a high-conductance Ca2+-activated potassium (MaxiK) channel in LPS signaling in macrophages. Corresponding to their biological activity, endotoxins activate a MaxiK channel as shown in outside-out patch-clamp experiments. LPS antagonists and anti-CD14 antibodies inhibit the LPS-induced activation of the channel. Blocking of the channel by specific channel blockers in macrophage cultures leads to inhibition of cytokine mRNA production. In particular, this result implies that there is no other independent transmembrane signaling pathway operative in macrophages. A shift of the molecular conformation of an a priori antagonistic lipid A from a cylindrical to a conical shape by adding the membrane-active compound chlorpromazine increases the activity of the MaxiK channel and the biological activity of the lipid A. We conclude that the activation of the MaxiK channel is a very early step in LPS-induced signaling in macrophages.

[Identification of wear particles of joint prostheses in tissues using laser microprobe mass analysis (LAMMA)]. / Identifizierung von Abriebpartikeln der Gelenkendoprothesen im Gewebe mit Hilfe der Laser-Mikrosonden-Massenanalyse (LAMMA).

The definite identification of wear particles from joint prostheses is of great importance for the development of joint replacement, as the type and quantity of different wear particles gives information on the wear resistance of implant materials. From the types of prostheses nowadays in use polyethylene wear of the sockets, bone cement wear, metallic and ceramic wear can be generated. Whereas polyethylene wear can be easily identified by its bright luminescence in polarized light and its characteristic configuration, the distinction of the small granular wear particles of the bone cement, metal and ceramic by light microscope is difficult. The laser microprobe mass analysis (LAMMA) is a method, which allows the analysis of single light microscopically detectable wear particles in tissues. Not only contrast medium particles of the bone cements (zirconium oxide or barium sulfate) but also metallic and aluminum oxide particles could be definitely identified within the pseudocapsules as well as in regional lymph nodes by LAMMA-analysis, whereby the bone cement wear predominated. In addition, the distinction between organic substances (as blood degradation products), which may appear similar to wear particles in configuration and colour, and the foreign material is also possible with this method.

Dual role of lipopolysaccharide (LPS)-binding protein in neutralization of LPS and enhancement of LPS-induced activation of mononuclear cells.

The lipopolysaccharide (LPS)-binding protein (LBP) has a concentration-dependent dual role in the pathogenesis of gram-negative sepsis: low concentrations of LBP enhance the LPS-induced activation of mononuclear cells (MNC), whereas the acute-phase rise in LBP concentrations inhibits LPS-induced cellular stimulation. In stimulation experiments, we have found that LBP mediates the LPS-induced cytokine release from MNC even under serum-free conditions. In biophysical experiments we demonstrated that LBP binds and intercalates into lipid membranes, amplified by negative charges of the latter, and that intercalated LBP can mediate the CD14-independent intercalation of LPS into membranes in a lipid-specific and temperature-dependent manner. In contrast, prior complexation of LBP and LPS inhibited binding of these complexes to membranes due to different binding of LBP to LPS or phospholipids. This results in a neutralization of LPS and, therefore, to a reduced production of tumor necrosis factor by MNC. We propose that LBP is not only present as a soluble protein in the serum but may also be incorporated as a transmembrane protein in the cytoplasmic membrane of MNC and that the interaction of LPS with membrane-associated LBP may be an important step in LBP-mediated activation of MNC, whereas LBP-LPS complexation in the serum leads to a neutralization of LPS.

Influence of acyl chain fluidity on the lipopolysaccharide-induced activation of complement.

Lipopolysaccharides (LPSs, endotoxins) are the major amphiphilic constituents of the outer leaflet of the outer membrane of Gram-negative bacteria. They are known to activate the complement cascade to form lytic membrane pores. Here, we study the influence of the fluidity of the acyl chains of LPSs and lipid As on the formation of lytic pores. To this end, we have performed electrical measurements on asymmetric planar endotoxin/phospholipid bilayers as a reconstitution model of the outer membrane using two deep rough mutant LPSs (from Escherichia coli strains WBB01 and WBB25) and two lipid As (from E. coli WBB25 and Rhodobacter sphaeroides). The two LPSs and the two lipid As each differ in their acylation pattern which is correlated with the fluidity. The addition of human serum to the endotoxin side of the bilayers led to the formation of membrane pores, and pore formation correlated in each case with acyl chain fluidity, i.e. time required for the first lytic pore to be formed was shorter for the more fluid endotoxin. Furthermore, in the case of LPSs, the activation rate was higher for the more fluid membrane and the respective bacteria had a higher susceptibility to the growth inhibitory action of serum.

An investigation into the membrane-interactive potential of the Escherichia coli KpsE C-terminus.

Membrane binding via C-terminal amphiphilic alpha-helical structure is a novel anchoring mechanism, which has been characterised in a number of prokaryotic carboxypeptidases. Here, we have used graphical and DWIH analyses to ascertain if a similar anchoring mechanism may be utilised by the Escherichia coli KpsE protein in its binding to the periplasmic face of the inner membrane. The results of these analyses have been compared to those obtained for similar analyses of the C-terminal sequences of E. coli penicillin-binding proteins (PBPs) PBP5 and PBP6 which, are known to function as amphiphilic alpha-helical membrane anchors, and of melittin, a known membrane-interactive toxin. We have also used FTIR spectroscopy and lipid phase transition temperature analysis to investigate the interaction of a peptide homologue of KpsE C-terminal region with membrane lipid. Our results suggest that the KpsE C-terminal sequence has the potential to form an amphiphilic alpha-helix and that this alpha-helix could feature in the membrane binding of the protein.

Bundle sheath cells of small veins in maize leaves are the location of uptake from the xylem.

Rb(+) as a tracer for K(+) was used to test the hypothesis that uptake of K(+) from xylem vessels of small veins into the symplast of maize leaves occurs at the xylem/bundle sheath cell interface. 22.5 min after immersing cut leaves into 20 mM RbCl+1 mM KCl, Rb(+) appeared in the cells of the leaves. Sections of these leaves were freeze-dried. In cryo-thin sections (5 microm), (85)Rb(+) and (41)K(+) content was determined by laser microprobe mass analysis with a large resolution of about 1 microm. Determining the ratio of (85)Rb(+) to (41)K(+) in the cell walls and cytosols of bundle sheath cells, mesophyll cells, and in the cells between the xylem elements resulted in the following picture: In small veins, Rb(+) entered the symplast directly at the xylem/bundle sheath cell interface.

Cytoplasmic membrane of a sensitive yeast is a primary target for Cryptococcus humicola mycocidal compound (microcin).

A basidiomycetous yeast strain, Cryptococcus humicola 9-6, secretes a mycocidal compound (microcin) which is lethal for many yeasts. In this study a new protocol for microcin purification has been developed, and TLC-purity product was obtained. Using fluorescein as a pH-sensitive probe it was found that microcin treatment of Cryptococcus terreus, a model microcin-sensitive yeast, immediately caused transient alkalization followed by acidification of the cells' cytoplasm. Upon completion of this process, endogenous respiration as well as activity of unspecific esterases were inhibited, and alterations in cell wall and/or capsule started. Microcin was shown to make the cells leaky for intracellular ATP. The mycocidal effect of microcin did not depend on the cell cycle phase of Cr. terreus. Based on these observations and on electrical measurements on planar phospholipid bilayers, which indicated a microcin-induced membrane permeabilization, it is suggested that the cytoplasmic membrane of the sensitive yeast is a primary target of microcin action. The conjectured mode of microcin action involves gradual increase of the cytoplasmic membrane's unspecific permeability. Intracellular ion homeostasis changes induced by microcin are considered to be the main cause of enzyme inhibition, alterations in the outer layers of the cell envelope and, finally, division arrest.

Physico-chemical analysis of lipid A fractions of lipopolysaccharide from Erwinia carotovora in relation to bioactivity.

Highly purified bisphosphoryl, monophosphoryl and dephosphoryl lipids A from Erwinia carotovora with different acylation patterns were characterized physico-chemically. Applying matrix assisted laser desorption/ionization mass spectrometry, the purity of the lipid A fractions was determined, and from monolayer measurements the molecular space requirement was estimated. Fourier transform infrared spectroscopy allowed the elucidation of the gel to liquid crystalline phase transition of the acyl chains as well as the determination of the tilt angle of the diglucosamine backbone with respect to the acyl chain direction applying dichroitic measurements with attenuated total reflectance. With synchrotron radiation small-angle X-ray diffraction the supramolecular aggregate structure was determined, and with fluorescence resonance energy transfer spectroscopy the lipopolysaccharide binding protein induced intercalation of lipid A into a phospholipid matrix corresponding to that of the macrophage membrane was investigated. From the results, a clear dependence of the physico-chemical parameters on the particular lipid A structure can be followed. Furthermore, these parameters correlate well with the biological activities of the various lipids A as deduced from their ability to induce biological activity (Limulus assay and cytokine induction in mononuclear cells). These results contribute to a closer interpretation of the physico-chemical prerequisites for endotoxic activity as found for enterobacterial lipid A.

Interaction between lipopolysaccharide (LPS), LPS-binding protein (LBP), and planar membranes.

The mechanism of interaction of the lipopolysaccharide (LPS)-binding protein, LBP, with differently composed symmetric and asymmetric planar lipid bilayers was investigated in electrical measurements (membrane current, potential, capacitance). From a change of the inner membrane potential difference, binding of LBP to membranes was deduced. After addition of LBP to one side of the membrane, binding of anti-LBP antibodies and LPS to LBP on both sides of the bilayer was observed. Effects resulting from an interaction of anti-LBP antiserum with membrane-bound LBP depend on the side of addition of the antiserum, indicating a directed intercalation of LBP into the membrane. Addition of LPS to the same side as LBP may induce a change of the conformation of LBP or its orientation in the membrane. Based on these observations, we propose that LBP intercalates in a directed orientation into negatively-charged membranes and assumes a transmembrane configuration. Moreover, pre-incubated complexes of LPS and LBP do not interact with membranes. These experiments show that reconstituted planar membranes are a suitable tool for investigations of the interaction of non pore-forming proteins that are involved in signal transduction.

Interaction of CAP18-derived peptides with membranes made from endotoxins or phospholipids.

Antimicrobial peptides with alpha-helical structures and positive net charges are in the focus of interest with regard to the development of new antibiotic agents, in particular against Gram-negative bacteria. Interaction between seven polycationic alpha-helical CAP18-derived peptides and different types of artificial membranes composed of phosphatidylcholine or lipopolysaccharide of the Gram-negative bacterium Escherichia coli were investigated using different biophysical techniques. Results obtained from fluorescence energy transfer spectroscopy with liposomes, monolayer measurements on a Langmuir trough, and electrophysiological measurements on planar reconstituted asymmetric bilayer membranes including the lipid matrix of the outer membrane of E. coli were correlated, and these data were, furthermore, correlated with structural parameters of the peptides (net charge, alpha-helical content, hydrophobic moment, and hydrophobicity). All peptides induced current fluctuations in planar membranes due to the formation of transient lesions above a peptide- and lipid-specific minimal clamp voltage. Antibacterial activity was exhibited only by those peptides that induced lesion formation in the reconstituted outer membrane at clamp voltages below the transmembrane potential of the natural membrane. Thus, we propose that the physicochemical properties of both the peptides as well as of the target membranes are important for antibacterial activity.

New insights into endotoxin-induced activation of macrophages: involvement of a K+ channel in transmembrane signaling.

LPS (endotoxins) activate cells of the human immune system, among which are monocytes and macrophages, to produce endogenous mediators. These regulate the immune response, but may also cause severe harm leading to septic shock. The activation of monocytes/macrophages by LPS is mediated by a membrane-bound LPS receptor, mCD14. As mCD14 lacks a transmembrane domain, a further protein is required for the signal transducing step to the cell interior. Here we show, using excised outside-out membrane patches, that activation of a high-conductance Ca(2+)- and voltage-dependent potassium channel is an early step in the transmembrane signal transduction in macrophages. The channel is activated by endotoxically active LPS in a dose-dependent manner. Channel activation can be completely inhibited by LPS antagonists and by anti-CD14 Abs. Activation of the channel is essential for LPS-induced cytokine production as shown by its inhibition by selective K(+) channel blockers.

Thermotropic and lyotropic properties of long chain alkyl glycopyranosides. Part II. Disaccharide headgroups.

We have investigated the thermotropic and lyotropic properties of some long chain alkyl glycosides with disaccharide headgroups. The thermotropism was measured with polarising microscopy and additionally the lyotropism with the contact preparation method, Fourier-transform Infrared (FTIR) spectroscopy, X-ray diffraction and small angle neutron scattering. A broad thermotropic as well as lyotropic polymorphism was found. The compounds displayed thermotropic S(A) (lamellar) and cubic phases, and the investigation of the lyotropic phase behaviour led to the observation of inverted bicontinuous cubic V(II) phases, lamellar L(alpha) phases, normal bicontinuous cubic V(I) phases, normal columnar H(I) phases, normal discontinuous cubic I(I) phases and lyotropic cholesteric phases. The phases are discussed with respect to the chemical structures that have been varied systematically to derive structure-property relationships.

Physicochemical characteristics of triacyl lipid A partial structure OM-174 in relation to biological activity.

The triacylated lipid A partial structure OM-174 was characterized in detail using a variety of physical and biological techniques. OM-174 aggregates adopt the micellar HI structure. The temperature (Tc) of the gel to liquid-crystalline phase transition of the hydrocarbon chains is 0 degrees C, from which high fluidity of the acyl chains at 37 degrees C can be deduced. The molecular area of a single OM-174 molecule at a surface pressure of 30 mN x m-1 is 0.78 +/- 0.04 nm2. Conformational analyses, using IR spectroscopy, of the behavior of the various functional groups of OM-174 as compared with hexa-acyl lipid A suggest altered hydration of the phosphate charges and unusually strong hydration of the ester groups, which is probably related to the high accessibility of these groups to water in the micellar aggregate structure. OM-174 was shown to intercalate into a phospholipid membrane corresponding to the macrophage membrane within seconds in the presence, and within minutes to hours in the absence, of LPS-binding protein. In the Limulus amebocyte lysate assay, the triacyl lipid A is more than 105-fold less active than hexa-acyl lipid A, but only 10-fold less active in inducing IL-6 in human mononuclear cells, and equally active in inducing NO production in murine macrophages. These findings are used to explain the mechanism of the lipid A-induced cell activation.