Biomedical subjects

Andra B Schromm

Publications and source records attributed to Andra B Schromm.

10 recordsLinked to original sources

PubMed2006

Biophysical characterization of synthetic rhamnolipids.

Synthetic rhamnolipids, derived from a natural diacylated glycolipid, RL-2,2(14), produced by Burkholderia (Pseudomonas) plantarii, were analyzed biophysically. Changes in the chemical structures comprised variations in the length, the stereochemistry and numbers of the lipid chains, numbers of rhamnoses, and the occurrence of charged or neutral groups. As relevant biophysical parameters, the gel (beta) to liquid crystalline (alpha) phase behavior of the acyl chains of the rhamnoses, their three-dimensional supramolecular aggregate structure, and the ability of the compounds to intercalate into phospholipid liposomes in the absence and presence of lipopolysaccharide-binding protein were monitored. Their biological activities were examined as the ability to induce cytokines in human mononuclear cells and to induce chemiluminescence in monocytes. Depending on the particular chemical structures, the physicochemical parameters as well as the biological test systems show large variations. This relates to the acyl chain fluidity, aggregate structure, and intercalation ability, as well as the bioactivity. Most importantly, the data extend our conformational concept of endotoxicity, based on the intercalation of naturally originating amphiphilic virulence factors into membranes from immune cells. This 'endotoxin conformation', produced by amphiphilic molecules with a hydrophilic charged backbone and apolar hydrophobic moiety, and adopting inverted cubic aggregate structures, causes high mechanical stress in target immune cells on integral proteins, eventually leading to cell activation. Furthermore, biologically inactive rhamnolipids with lamellar aggregate structures antagonize the endotoxin-induced activity in a way similar to lipid A-derived antagonists.

Animals↗

PubMed2006

MaxiK blockade selectively inhibits the lipopolysaccharide-induced I kappa B-alpha /NF-kappa B signaling pathway in macrophages.

Macrophages have a pivotal function in innate immunity against bacterial infections. They are present in all body compartments and able to detect invading microorganisms with high sensitivity. LPS (endotoxin) of Gram-negative bacteria is among the most potent stimuli for macrophages and initiates a wide panel of cellular activation responses. The release of mediators such as TNF-alpha and ILs is essential for the initiation of a proinflammatory antibacterial response. Here, we show that blockade of the large-conductance Ca2+ -activated potassium channel MaxiK (BK) inhibited cytokine production from LPS-stimulated macrophages at the transcriptional level. This inhibitory effect of channel blockade was specific to stimulation with LPS and affected neither stimulation of macrophages with the cytokine TNF-alpha nor LPS-induced activation of cells that do not express MaxiK. Investigation of the upstream intracellular signaling pathways induced by LPS revealed that the blockade of MaxiK selectively inhibited signaling pathways leading to the activation of the transcription factor NF-kappaB and the MAPK p38, whereas activation of ERK was unaffected. We present data supporting that proximal regulation of the inhibitory factor IkappaB-alpha is critically involved in the observed inhibition of NF-kappaB translocation. Using alveolar macrophages from rats, we could show that the necessity of MaxiK function in activation of NF-kappaB and subsequent cytokine production is not restricted to in vitro-generated monocyte-derived macrophages but also can be observed in primary cells. Thus, MaxiK appears to be a central molecule in the NF-kappaB-dependent inflammatory response of macrophages to bacterial LPS.

Animals↗

PubMed2006

Cell activation of human macrophages by lipoteichoic acid is strongly attenuated by lipopolysaccharide-binding protein.

Lipoteichoic acid (LTA) represents immunostimulatory molecules expressed by Gram-positive bacteria. They activate the innate immune system via Toll-like receptors. We have investigated the role of serum proteins in activation of human macrophages by LTA from Staphylococcus aureus and found it to be strongly attenuated by serum. In contrast, the same cells showed a sensitive response to LTA and a significantly enhanced production of tumor necrosis factor alpha under serum-free conditions. We show that LTA interacts with the serum protein lipopolysaccharide-binding protein (LBP) and inhibits the integration of LBP into phospholipid membranes, indicating the formation of complexes of LTA and soluble LBP. The addition of recombinant human LBP to serum-free medium inhibited the production of tumor necrosis factor alpha and interleukins 6 and 8 after stimulation of human macrophages with LTA in a dose-dependent manner. Using anti-LBP antibodies, this inhibitory effect could be attributed to soluble LBP, whereas LBP in its recently described transmembrane configuration did not modulate cell activation. Also, using primary alveolar macrophages from rats, we show a sensitive cytokine response to LTA under serum-free culture conditions that was strongly attenuated in the presence of serum. In summary, our data suggest that innate immune recognition of LTA is organ-specific with negative regulation by LBP in serum-containing compartments and sensitive recognition in serum-free compartments like the lung.

Acute-Phase Proteins↗

PubMed2006

Cell activation by ligands of the toll-like receptor and interleukin-1 receptor family depends on the function of the large-conductance potassium channel MaxiK in human macrophages.

Performing patch-clamp experiments on human macrophages, we show that the K(+) channel MaxiK is activated by lipopolysaccharide, peptidoglycan, and interleukin-1. Cytokine production initiated by several Toll-like receptor (TLR) ligands and by interleukin-1 is inhibited by MaxiK blockade. This provides evidence for functional association of the MaxiK channel and TLR signaling complexes.

Cell Line↗

PubMed2005

Divalent cations affect chain mobility and aggregate structure of lipopolysaccharide from Salmonella minnesota reflected in a decrease of its biological activity.

The physicochemical properties and biological activities of rough mutant lipopolysaccharides Re (LPS Re) as preformed divalent cation (Mg2+, Ca2+, Ba2+) salt form or as natural or triethylamine (Ten+)-salt form under the influence of externally added divalent cations were investigated using complementary methods: Differential scanning calorimetry (DSC) and Fourier-transform infrared spectroscopic (FT-IR) measurements for the beta <--> alpha gel to liquid crystalline phase behaviour of the acyl chains of LPS, synchrotron radiation X-ray diffraction studies for their aggregate structures, electron density calculations of the LPS bilayer systems, and LPS-induced cytokine (interleukin-6) production in human mononuclear cells. The divalent cation salt forms of LPS exhibit considerable changes in physicochemical parameters such as acyl chain mobility and aggregate structures as compared to the natural or monovalent cation salt forms. Concomitantly, the biological activity was much lower in particular for the Ca2+- and Ba2+-salt forms. This decrease in activity results mainly from the conversion of the unilamellar/cubic aggregate structure of LPS into a multilamellar one. The reduced activity also clearly correlates with the higher order--lower mobility--of the lipid A acyl chains. Both effects can be understood by an impediment of the interactions of LPS with binding proteins such as lipopolysaccharide-binding protein (LBP) and CD14 due to the action of the divalent cations.

Calorimetry, Differential Scanning↗

PubMed2005

Phospholipids inhibit lipopolysaccharide (LPS)-induced cell activation: a role for LPS-binding protein.

The inhibition of LPS-induced cell activation by specific antagonists is a long-known phenomenon; however, the underlying mechanisms are still poorly understood. It is commonly accepted that the membrane-bound receptors mCD14 and TLR4 are involved in the activation of mononuclear cells by LPS and that activation may be enhanced by soluble LPS-binding protein (LBP). Hexaacylated Escherichia coli lipid A has the highest cytokine-inducing capacity, whereas lipid A with four fatty acids (precursor IVa, synthetic compound 406) is endotoxically inactive, but expresses antagonistic activity against active LPS. Seeking to unravel basic molecular principles underlying antagonism, we investigated phospholipids with structural similarity to compound 406 with respect to their antagonistic activity. The tetraacylated diphosphatidylglycerol (cardiolipin, CL) exhibits high structural similarity to 406, and our experiments showed that CL strongly inhibited LPS-induced TNF-alpha release when added to the cells before stimulation or as a CL/LPS mixture. Also negatively charged and to a lesser degree zwitterionic diacyl phospholipids inhibited LPS-induced cytokine production. Using Abs against LBP, we could show that the activation of cells by LPS was dependent on the presence of cell-associated LBP, thus making LBP a possible target for the antagonistic action of phospholipids. In experiments investigating the LBP-mediated intercalation of LPS and phospholipids into phospholipid liposomes mimicking the macrophage membrane, we could show that preincubation of soluble LBP with phospholipids leads to a significant reduction of LPS intercalation. In summary, we show that LBP is a target for the inhibitory function of phospholipids.

Acute-Phase Proteins↗

PubMed2005

Physicochemical characterization of carboxymethyl lipid A derivatives in relation to biological activity.

Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria belongs to the most potent activators of the mammalian immune system. Its lipid moiety, lipid A, the 'endotoxic principle' of LPS, carries two negatively charged phosphate groups and six acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506). Tetraacyl lipid A (precursor IVa or synthetic 406), which lacks the two hydroxylated acyl chains, is agonistically completely inactive, but is a strong antagonist to bioactive LPS when administered to the cells before LPS addition. The two negative charges of lipid A, represented by the two phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups. We hypothesized that the phosphate groups could be substituted by other negatively charged groups without changing the endotoxic properties of lipid A. To test this hypothesis, we synthesized carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-CM-506) and of tetraacyl lipid A (Bis-CM-406) and correlated their physicochemical with their endotoxic properties. We found that, similarly to compounds 506 and 406, also for their carboxymethyl derivatives a particular molecular ('endotoxic') conformation and with that, a particular aggregate structure is a prerequisite for high cytokine-inducing capacity and antagonistic activity, respectively. In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3-deoxy-D-manno-oct-2-ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed. These data allow a better understanding of endotoxic activity and its structural prerequisites.

Antibodies, Monoclonal↗

PubMed2004

Investigation into the interaction of the bacterial protease OmpT with outer membrane lipids and biological activity of OmpT:lipopolysaccharide complexes.

Outer-membrane proteases T (OmpT) are important defence molecules of Gram-negative bacteria such as Escherichia coli found in particular in clinical isolates. We studied the interaction of OmpT with the membrane-forming lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) from the inner leaflet and lipopolysaccharide (LPS) from the outer leaflet of the outer membrane. These investigations comprise functional aspects of the protein-lipid interaction mimicking the outer-membrane system as well as the bioactivity of LPS:OmpT complexes in the infected host after release from the bacterial surface. The molecular interaction of the lipids PE, PG, and LPS with OmpT was investigated by analysing molecular groups in the lipids originating from the apolar region (methylene groups), the interface region (ester), and the polar region (phosphates), and by analysing the acyl-chain melting-phase behaviour of the lipids. The activity of OmpT and LPS:OmpT complexes was investigated in biological test systems (human mononuclear cells and Limulus amoebocyte lysate assay) and with phospholipid model membranes. The results show a strong influence of OmpT on the mobility of the lipids leading to a considerable fluidization of the acyl chains of the phospholipids as well as LPS, and a rigidification of the phospholipid, but not LPS head groups. From this, a dominant role of the protein on the function of the outer membrane can be deduced. OmpT released from the outer membrane still contains slight contaminations of LPS, but its strong cytokine-inducing ability in mononuclear cells, which does not depend on the Toll-like receptors 2 and 4, indicates an LPS-independent mechanism of cell activation. This might be of general importance for infections induced by Gram-negative bacteria.

Animals↗

PubMed2004

Aggregates are the biologically active units of endotoxin.

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta- and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity.

Animals↗

PubMed2003

The generalized endotoxic principle.

Bacterial lipopolysaccharides (endotoxins, LPS) belong to the most potent immunostimulators in mammals. The endotoxic principle of LPS is located in its lipid A moiety, which for Escherichia coli-type LPS consists of a hexaacylated diphosphoryl diglucosamine backbone. This lipid A adopts a cubic inverted aggregate structure from which a conical shape of the molecule can be deduced, whereas the tetraacyl lipid A precursor IVa adopts a cylindrical shape and is endotoxically inactive, but antagonizes active LPS. We hypothesize that non-lipid A amphiphiles with similar physicochemical properties of amphiphilicity, charge, and shape, might mimic the respective lipid A. To test this hypothesis, phospholipid-like amphiphiles with six acyl chains attached to a bisphosphorylated serine-like backbone of varying length replacing the diglucosamine backbone were synthesized. The compound with a short backbone fulfills all criteria of an endotoxic agonist, and that with long backbone fulfills those of an antagonist. This holds true for the human as well as for the murine system. Interestingly, these compounds are inactive in the Limulus amebocyte lysate test which is specific for LPS diglucosamine backbone. These results define a general endotoxic principle and, furthermore, provide new insights into an understanding of early steps of endotoxin action.

Adult↗