Isolation and characterization of a complement-activating lipid extracted from human atherosclerotic lesions.

The major characteristics of human atherosclerotic lesions are similar to those of a chronic inflammatory reaction, namely fibrosis, mesenchymal cell proliferation, the presence of resident macrophages, and cell necrosis. Atherosclerosis exhibits in addition the feature of lipid (mainly cholesterol) accumulation. The results of the present report demonstrate that a specific cholesterol-containing lipid particle present in human atherosclerotic lesions activates the complement system to completion. Thus, lipid could represent a stimulatory factor for the inflammatory reaction, whose underlying mechanistic basis may be, at least in part, complement activation. The complement-activating lipid was purified from saline extracts of aortic atherosclerotic lesions by sucrose density gradient centrifugation followed by molecular sieve chromatography on Sepharose 2B. It contained little protein other than albumin, was 100-500 nm in size, exhibited an unesterified to total cholesterol ratio of 0.58 and an unesterified cholesterol to phospholipid ratio of 1.2. The lipid, termed lesion lipid complement (LCA), activated the alternative pathway of complement in a dose-dependent manner. Lesion-extracted low density lipoprotein (LDL) obtained during the purification procedure failed to activate complement. Specific generation of C3a desArg and C5b-9 by LCA indicated C3/C5 convertase formation with activation proceeding to completion. Biochemical and electron microscopic evaluations revealed that much of the C5b-9 present in atherosclerotic lesions is membraneous, rather than fluid phase SC5b-9. The observations reported herein establish a link between lipid insudation and inflammation in atherosclerotic lesions via the mechanism of complement activation.

Phase-variable expression of lipopolysaccharide contributes to the virulence of legionella pneumophila.

With the aid of monoclonal antibody (mAb) 2625, raised against the lipopolysaccharide (LPS) of Legionella pneumophila serogroup 1, subgroup OLDA, we isolated mutant 811 from the virulent wild-type strain RC1. This mutant was not reactive with mAb 2625 and exhibited an unstable phenotype, since we observed an in vitro and in vivo switch of mutant 811 to the mAb 2625-positive phenotype, thus restoring the wild-type LPS. Bactericidal assays revealed that mutant 811 was lysed by serum complement components, whereas the parental strain RC1 was almost serum resistant. Moreover, mutant 811 was not able to replicate intracellularly in macrophage-like cell line HL-60. In the guinea pig animal model, mutant 811 exhibited significantly reduced ability to replicate. Among recovered bacteria, mAb 2625-positive revertants were increased by fourfold. The relevance of LPS phase switch for pathogenesis of Legionella infection was further corroborated by the observation that 5% of the bacteria recovered from the lungs of guinea pigs infected with the wild-type strain RC1 were negative for mAb 2625 binding. These findings strongly indicate that under in vivo conditions switching between two LPS phenotypes occurs and may promote adaptation and replication of L. pneumophila. This is the first description of phase-variable expression of Legionella LPS.

Regulation of fetal hemoglobin expression during hematopoietic stem cell development and its importance in bone metabolism and osteoporosis.

We have shown that an altered tissue redox environment in mice lacking either murine beta Hemoglobin major (HgbßmaKO) or minor (HgbßmiKO) regulates inflammation. The REDOX environment in marrow stem cell niches also control differentiation pathways. We investigated osteoclastogenesis (OC)/osteoblastogenesis (OB), in bone cultures derived from untreated or FSLE-treated WT, HgbßmaKO or HgbßmiKO mice. Marrow mesenchymal cells from 10d pre-cultures were incubated on an osteogenic matrix for 21d prior to analysis of inflammatory cytokine release into culture supernatants, and relative OC:OB using (TRAP:BSP, RANKL:OPG) mRNA expression ratios and TRAP or Von Kossa staining. Cells from WT and HgbßmaKO mice show decreased IL-1ß,TNFα and IL-6 production and enhanced osteoblastogenesis with altered mRNA expression ratios and increased bone nodules (Von Kossa staining) in vitro after in vivo stimulation of mRNA expression of fetal Hgb genes (Hgbε and Hgbßmi) by a fetal liver extract (FSLE). Marrow from HgbßmiKO showed enhanced cytokine release and preferential enhanced osteoclastogenesis relative to similar cells from WT or HgbßmaKO mice, with no increased osteoblastogenesis after mouse treatment with FSLE. Pre-treatment of WT or HgbßmaKO, but not HgbßmiKO mice, with other molecules (rapamycin; hydroxyurea) which increase expression of fetal Hgb genes also augmented osteoblastogenesis and decreased cytokine production in cells differentiating in vitro. Infusion of rabbit anti- Hgbε or anti- Hgbßmi, but not anti-Hgbα or anti- Hgbßma into WT mice from day 13 gestation for 3 weeks led to attenuated osteoblastogenesis in cultured cells. We conclude that increased fetal hemoglobin expression, or use of agents which improve fetal hemoglobin expression, increases osteoblast bone differentiation in association with decreased inflammatory cytokine release.

An alteration in the levels of populations of CD4+ Treg is in part responsible for altered cytokine production by cells of aged mice which follows injection with a fetal liver extract.

We have shown previously that a fetal sheep liver extract (FSLE) containing significant quantities of fetal ovine gamma globin chain (Hbgamma) and LPS injected into aged (>20 months) mice could reverse the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFNgamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. The mechanism(s) behind this change in cytokine production were not previously investigated. We report below that aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+) Treg and so-called Tr3 (CD4(+)TGFbeta(+)), and that their number/function is restored to levels seen in control (8-week-old) mice by FSLE. In addition, on a per cell basis, CD4(+)CD25(-)Treg from aged mice were >4-fold more effective in suppression of proliferation and IL-2 production from ConA-activated lymphoid cells of a pool of CD4(+)CD25(-)T cells from 8-week-old mice than similar cells from young animals, and this suppression by CD25(-)T cells was also ameliorated following FSLE treatment. Infusion of anti-TGFbeta and anti-IL-10 antibodies in vivo altered Treg development following FSLE treatment, and attenuated FSLE-induced alterations in cytokine production profiles.

An altered REDOX environment, assisted by over-expression of fetal hemoglobins, protects from inflammatory colitis and reduces inflammatory cytokine expression.

C5BL/6 female mice receiving dextran sodium sulfate in their drinking water develop an acute inflammatory colitis within 7d, with weight loss, histopathologic signs of inflammation, and colonic expression of inflammatory cytokines. In previous studies we have reported that increased inflammatory cytokine expression in aged mice can be attenuated by oral gavage of a crude fetal extract containing glutathione (GSH), MPLA and fetal hemoglobin, or more specifically by injection of a combination of these purified reagents. We speculated that this combination led to an altered tissue redox environment in which the immune response developed, thus regulating inflammation. Accordingly, we used wild-type (WT) C57BL/6 mice, or mice lacking either murine beta Hemoglobin major (HgbßmaKO) or minor (HgbßmiKO) as recipients of DSS in their drinking water, and followed development of colitis both clinically and by inflammatory cytokine production, before/after oral treatment of mice with a crude fetal liver extract. Mice lacking an intact fetal hemoglobin chain (HgbßmiKO) developed severe colitis, with enhanced colonic expression of inflammatory cytokines, which could not be rescued by extract, unlike WT and HgbßmaKO animals. Moreover, disease in both WT and HgbßmaKO animals could also be attenuated by exposure to 5-hydroxymethyl furfural (5HMF), hydroxyurea or rapamycin. The former has been used as an alternative means of stabilizing the conformation of adult hemoglobin in a manner which mimicks the oxygen-affinity of fetal hemoglobin, while we show that both hydroxyurea and rapamycin augment expression of murine fetal hemoglobin chains. Our data suggests there may be a clinical value in exploring agents which alter local REDOX environments as an adjunctive treatment for colitis and attenuating inflammatory cytokine production.

Role of MIF and glutathione, in association with fetal ovine globin chain (Hbgamma) and LPS, in induction of TNFalpha from cells of young and aged mice, and PBL from healthy human populations.

Previous reports from our group have established that the fetal ovine gamma globin chain (Hbgamma) and LPS can synergize in the induction of pro-inflammatory cytokines, especially TNFalpha, from mouse and human leukocytes. A fetal sheep liver extract (FSLE) which was observed to have marked immunoregulatory properties in vivo and in vitro had independently been observed to contain significant amounts of each of these molecules. However, the biological activity of this extract (hereafter FSLE) was not explained solely by its content of Hbgamma and LPS, and independent analysis confirmed also the presence of migration inhibitory factor, MIF, and glutathione in FSLE. We have investigated whether MIF and the cellular anti-oxidant glutathione can further synergize with Hbgamma and LPS in TNFalpha induction from human cells in vitro, and mouse cells activated in vivo/in vitro. Our data show that indeed there is evidence for such a synergy. Treatment or mouse cells with FSLE produced an enhanced TNFalpha production which could be inhibited independently both by anti-Hbgamma and by anti-MIF, and optimally by a combination of these reagents.

Immunopathogenesis of atherosclerosis: endotoxin accelerates atherosclerosis in rabbits on hypercholesterolemic diet.

BACKGROUND: On the basis of our concept that atherosclerosis has an immunopathological background, we tested whether activation of the innate immune system influences its progression. METHODS AND RESULTS: Hypercholesterolemic (0.5% wt/wt diet) rabbits received either repeated intravenous injections of endotoxin (Escherichia coli lipopolysaccharide 1.25 to 2.5 microg, once per week) or a self-limiting cutaneous Staphylococcus aureus infection with or without a quinolone antibiotic. Measured laboratory parameters, including LDL and HDL cholesterols, were similar in the different groups of hypercholesterolemic animals. All endotoxin-treated animals developed transient episodes of fever after endotoxin administration. The extent of atherosclerosis was evaluated by computer-assisted morphometry in the aortas en face (Sudan IV) and by histology at 8 weeks after start of the experiments. Endotoxin-treated animals exhibited significantly accelerated atherosclerosis compared with control animals (141+/-38 versus 45+/-16 mm(3) total lesion volume, n=7 to 9 rabbits each, P<0.001). CONCLUSIONS: Nonspecific stimulation of the innate immune system accelerates cholesterol-induced atherosclerosis. These data support the concept that atherosclerosis has an immunopathological component and render it improbable that a single infectious agent should assume particular importance in its initiation or progression.

Analysis of interaction of cloned human and/or sheep fetal hemoglobin gamma-chain and LPS in augmenting induction of inflammatory cytokine production in vivo and in vitro.

We have reported earlier that purified preparations of sheep fetal hemoglobin, but not adult hemoglobin, in concert with non-stimulatory doses of lipopolysaccharide (LPS) (lipid A), act cooperatively to regulate in vitro production of a number of cytokines, including TNFalpha, TGFbeta and IL-6 from murine and human leukocytes. Following in vivo treatment of mice with the same combination of hemoglobin and LPS, harvested spleen or peritoneal cells showed a similar augmented capacity to release these cytokines into culture supernatants. We report below that genetically cloned gamma-chain of human or sheep fetal hemoglobin, but not cloned alpha- or beta-chains, can produce this cooperative effect, as indeed can HPLC purified, heme-free, gamma-chains derived from cord blood fetal hemoglobin, and that purified haptoglobin completely abolishes the cooperative interaction.

Structural and biological characterisation of a novel tetra-acyl lipid A from Escherichia coli F515 lipopolysaccharide acting as endotoxin antagonist in human monocytes.

We here report on the structural analysis of a novel tetra-acyl lipid A (LA (tetra) ) isolated from Escherichia coli deep rough (Re)-mutant strain F515. In addition to the biologically active hexa-acyl E. coli-type lipid A (compound 506), this incompletely acylated lipid A was found to be also present in the native LPS. Its structure was studied without further derivatisation by chemical analysis, matrix-assisted laser desorption/ionization mass spectrometry, and one- and two-dimensional (1)H- and (13)C-NMR spectroscopy. It was found to be structurally distinct from the tetra-acyl lipid A biosynthetic precursor Ia (compound 406) in lacking the primary (R)-3-hydroxytetradecanoic acid 14:0(3-OH) in position 3' ester-linked to the 'non-reducing' glucosamine (GlcN II). The hydroxyl group at the (R)-3-hydroxytetradecanoic acid attached to position 2' of GlcN II was found to be substituted by dodecanoic acid (12:0), thus forming a dodecanoyloxytetradecanoyl residue 14:0[3-O(12:0)]. The acylation pattern at the 'reducing' GlcN I was identical to that of compound 406 in having two primary (R)-3-hydroxy tetradecanoic acid residues [14:0(3-OH)] attached to positions 3 (ester-linked) and 2 (amide-linked), respectively. In human mononuclear cells (hMNC) the new LA (tetra) antagonized LPS-induced release of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in a dose-dependent manner with identical antagonistic potency as compared with compound 406. Also like compound 406, it was found to be an agonist in murine macrophage-like J774.1 cells.

Chemical structure of glycosphingolipids isolated from Sphingomonas paucimobilis.

Two novel glycosphingolipids were isolated from Sphingomonas paucimobilis and their structures were completely elucidated. The glycosyl portion of the glycosphingolipid consists of an alpha-D-Manp-[1----2)-alpha-D-Galp-(1----6)-alpha-D-GlcpN-(1 ----4)-alpha-D- GlcpA-R tetrasaccharide. The hydrophobic residue R was found to be heterogeneous with respect to the dihydrosphingosine residue. Erythro-1,3-dihydroxy-2-amino-octadecane and erythro-1,3-dihydroxy-2-amino-cis-13,14-methyleneoctadecane were identified in comparable amounts. Both dihydrosphingosine derivatives were quantitatively substituted by an (S)-2-hydroxymyristic acid in amide linkage.

Functional characterization of sphingolipid C4-hydroxylase genes from Arabidopsis thaliana.

In the genome of Arabidopsis thaliana, two genes were identified encoding isoenzymes for C4-hydroxylation of long chain bases (LCB) in plant sphingolipids. Both predicted proteins consist of 258 amino acid residues (77% identity) which show sequence similarity to di-iron-binding enzymes, such as Sur2p and Erg3p from yeast, involved in oxygen-dependent lipid modifications. Heterologous expression of these genes in a yeast sur2Delta-null mutant lacking C4-LCB hydroxylation resulted in the formation of D-ribo-C(18)- and -C(20)-phytosphinganine. The identity and stereochemical configuration of the isolated trihydroxybases was confirmed by electrospray ionization-mass spectroscopy, gas-liquid chromatography-mass spectrometry and 1H-nuclear magnetic resonance spectroscopy. These results represent the first functional identification of SUR2 genes from plants as well as from any organism other than yeast.

Lipopolysaccharide and peptidoglycan: CD14-dependent bacterial inducers of inflammation.

Surface structures of bacteria contribute to the microbial pathogenic potential and are capable of causing local and generalized inflammatory reactions. Among these factors, endotoxin and peptidoglycan are of particular medical importance. Both toxic bacterial polymers are now recognized to interact with the same cellular receptor, the CD14 molecule, which is expressed on different types of immune cells, in particular, monocytes/macrophages. The interaction between these bacterial activators and CD14 leads to the production of endogenous mediators such as tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-6, which are ultimately responsible for phlogistic responses. The fact that CD14 recognizes not only endotoxin and peptidoglycan but also other glycosyl-based microbial polymers suggests that this cellular surface molecule represents a lectin.

Bacterial endotoxin: molecular relationships between structure and activity.

The significance of endotoxins in bacterial infection and their role as bacterial surface antigens (O antigens) have stimulated investigations into their chemical nature and the mechanisms of their biologic action during the last few decades. This article summarizes some of the recent results and emphasizes structure-activity relationships.

Nourseothricin (streptothricin) inactivated by a plasmid pIE636 encoded acetyl transferase of Escherichia coli: location of the acetyl group.

Escherichia coli strains harbouring the plasmid pIE636 are able to synthesize acetylcoenzyme A: streptothricin acetyltransferase (ACSAT). The (enzymatic) N-acetylation of streptothricin F is known to contribute significantly towards the loss of antibacterial activity. 13C-NMR analysis of [14C]N-acetyl-labelled streptothricin F, produced by ACSAT-catalysed acetylation of streptothricin F and subsequent purification by various chromatographical steps, unequivocally revealed streptothricin F to be acetylated at the beta-amino group (C16) (and not at the epsilon-amino group (C19)).

The phosphocholine motif in membranes of Mycoplasma fermentans strains.

Mycoplasma fermentans strains differ in the profile of choline-containing phosphoglycolipids (PGL) present in their cell membrane. MfGL-II [Zähringer et al. (1997) J. Biol. Chem. 272, 26262-26270] was found to be the major PGL in most strains tested. However, in the pulmonary isolates, M52 and M39 the major choline-containing PGLs were MfGL-I [Matsuda et al. (1994) J. Biol. Chem. 269, 33123-33129] and MfEL, a unique choline-containing ether lipid recently identified by us [Wagner et al. (2000) Eur. J. Biochem. 267, 6276-6286]. MfGL-I, MfGL-II and MfEL were metabolically labeled by growing the cells with radioactive choline but only MfGL-I and MfGL-II [corrected] reacted with antiphosphocholine antibodies. All tested strains fused with Molt-3 cells at almost the same rate and to about the same extent and in all the strains membrane proteins that reacted with anti-phosphocholine antibodies were detected, indicating that some membrane proteins are decorated with phosphocholine moieties.

Long-chain alpha-hydroxy-(omega-1)-oxo fatty acids and alpha-hydroxy-1,omega-dioic fatty acids are cell wall constituents of Legionella (L. jordanis, L. maceachernii and L. micdadei).

Four long-chain fatty acids, 2-hydroxy-27-oxo-octacosanoic acid (n28:0(2-OH,27-oxo)), 2-hydroxy-29-oxo-triacontanoic acid (n30:0(2-OH,29-oxo)), 2-hydroxy-heptacosane-1,27-dioic acid (27:0(2-OH)-dioic) and 2-hydroxy-nonacosane-1,29-dioic acid (29:0(2-OH)-dioic) were identified by GLC-MS analysis in the phenol-chloroform-petroleum ether (PCP) extracts of Legionella jordanis, L. maceachernii and L. micdadei indicating that they are constituents of lipopolysaccharide. Moreover, five long-chain fatty acids (28:0(27-OH), 28:0(27-oxo), 30:0(29-oxo), 27:0-dioic and 29:0-dioic) previously identified in L. pneumophila (Moll, H. et al., FEMS Microbiol. Lett., 97 (1992), 1-6) were also found in these species. This is to our knowledge the first report on the existence of long chain 2-hydroxylated (omega-1)-oxo fatty acids and 2-hydroxylated 1,omega-dioic fatty acids.

Identification of 27-oxo-octacosanoic acid and heptacosane-1,27-dioic acid in Legionella pneumophila.

Two long-chain fatty acids, 27-oxo-octacosanoic acid (28:0(27-oxo)) and heptacosane-1,27-dioic acid (27:0-dioic) were identified for the first time in phenol-chloroform-petroleum ether extracts of Legionella pneumophila, indicating that they are constituents of lipopolysaccharide. The fatty acids were characterised by combined gas-liquid chromatography/mass spectrometry and proton nuclear magnetic resonance spectroscopy. Moreover, minor amounts of 29-oxo-triacontanoic (30:0(29-oxo)) acid and nonacosane-1,29-dioic acid (29:0-dioic) as well as 27-hydroxy-octacosanoic acid (28:0(27-OH)) were present in the phenol-chloroform-petroleum ether extract.

Choline deficiency induced by Mycoplasma fermentans enhances apoptosis of rat astrocytes.

A choline uptake system accumulating free choline in an energy-dependent process is described in Mycoplasma fermentans. The uptake system has a K(m) of 2.2x10(-5) M and a V(max) of 0.15 nmol 10 min(-1) mg(-1) cell protein and the choline incorporated could be recovered in the soluble fraction as free choline, phosphorylcholine and CDP-choline. Choline accumulation by M. fermentans resulted in a marked choline depletion of the growth medium. The choline depletion of an astrocyte cell culture induced by M. fermentans was associated with the apoptotic death of the cells. Apoptosis was not obtained with heat-inactivated mycoplasmas and could be reversed by the addition of free choline to the growth medium.

Antibody response to MfGL-II, a phosphocholine-containing major lipid of Mycoplasma fermentans membranes.

The choline-containing phosphoglycolipid, MfGL-II, is the major polar lipid of Mycoplasma fermentans PG18. Anti-MfGL-II antisera raised in rabbits using the purified MfGL-II as an immunogen were employed in immunogold electron microscopic and immunofluorescence studies showing that MfGL-II is uniformly distributed and exposed on the cell surface of M. fermentans cells. The specificity of the antibodies was determined by immunostaining of lipid extracts separated by thin layer chromatography. The antibodies recognize lipids specific to M. fermentans but did not cross-react with lipid extracts of M. penetrans, M. capricolum, M. gallisepticum or Acholeplasma laidlawii. As phosphocholine almost completely abolished antibody interaction with MfGL-II in an ELISA assay it is suggested that the anti-MfGL-II repertoire is composed primarily of anti-phosphocholine antibodies. The anti-MfGL-II antisera inhibit the attachment of M. fermentans to Molt-3 lymphocytes suggesting that MfGL-II plays a major role in M. fermentans-host cell interaction.

The significance of the hydrophilic backbone and the hydrophobic fatty acid regions of lipid A for macrophage binding and cytokine induction.

Natural partial structures of lipopolysaccharide (LPS) as well as synthetic analogues and derivatives of lipid A were compared with respect to inhibit the binding of 125I-labelled Re-chemotype LPS to mouse macrophage-like J774.1 cells and to induce cytokine-release in J774.1 cells. LPS, synthetic Escherichia coli-type lipid A (compound 506) and tetraacyl precursor Ia (compound 406) inhibited the binding of 125I-LPS to macrophage-like J774.1 cells and induced the release of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6). Deacylated R-chemotype LPS preparations were completely inactive in inhibiting binding and in inducing cytokine-release. Among tetraacyl compounds, the inhibition-capacity of LPS-binding was in decreasing order: PE-4 (alpha-phosphonooxyethyl analogue of 406) > 406 >> 404 (4'-monophosphoryl partial structure of 406) > 405 (1-monophosphoryl partial structure of 406). In the case of hexaacyl preparations, compounds 506, PE-1 (alpha-phosphonooxyethyl analogue of 506) and PE-2 (differing from PE-1 in having 14:0 at positions 2 and 3 of the reducing GlcN) inhibited LPS-binding and induced cytokine release equally well, whereas preparation PE-3 (differing from PE-2 in containing a beta-phosphonooxyethyl group) showed a substantially lower capacity in binding-inhibition and cytokine-induction. The conclusion is that chemical changes in the hydrophilic lipid A backbone reduce the capacity of lipid A to bind to cells, whereas the number of fatty acids determines the capacity of lipid A to activate cells. These results indicate that the bisphosphorylated hexosamine backbone of lipid A is essential for specific binding of LPS to macrophages and that the acylation pattern plays a critical role for LPS-promoted cell activation, i.e. cytokine induction.