Toll-like receptors in atherosclerosis.

At one time, atherosclerosis was thought to be a simple lipid storage disease. However, it is now recognized as a chronic and progressive inflammation of the arterial wall. Gene deletion experiments in murine models of atherosclerosis that reduce the inflammatory process also reduce disease severity. Identifying the initiators and mediators of that inflammation can provide promising avenues for prevention or therapy. Two prominent risk factors, hyperlipidaemia and infectious disease, point to innate immune mechanisms as potential contributors to proatherogenic inflammation. The TLRs (Toll-like receptors), pro-inflammatory sensors of pathogens, are potential links between inflammation, infectious disease and atherosclerosis. A mechanism for hyperlipidaemic initiation of sterile inflammation can be postulated because oxidized lipoproteins or their component oxidized lipids have been identified as TLR ligands. Moreover, infectious agents are correlated with atherosclerosis risk. We have identified a role for TLR2 in atherosclerosis in mice deficient in low-density lipoprotein receptor. We observed that proatherogenic TLR2 responses to unknown endogenous or unknown endemic exogenous agonists are mediated by non-BMDC (bone-marrow-derived cells), which can include endothelial cells. In contrast, the proatherogenic TLR2 responses to the defined synthetic exogenous agonist Pam3 CSK4 are mediated at least in part by BMDC, which can include lymphocytes, monocytes/macrophages and dendritic cells. TLR2-mediated cell activation in response to endogenous and exogenous agents is proatherogenic in hyperlipidaemic mice.

The toll of Toll-like receptors, especially toll-like receptor 2, on murine atherosclerosis.

At one time, atherosclerosis was thought to be a simple lipid storage disease. However, it is now recognized as a chronic and progressive inflammation of the arterial wall. Gene deletion experiments in murine models of atherosclerosis that reduce the inflammatory process also reduce disease severity. Identifying the initiators and mediators of that inflammation can provide promising avenues for prevention or therapy. Two prominent risk factors, hyperlipidemia and infectious disease, point to innate immune mechanisms as potential contributors to proatherogenic inflammation. The Toll-like receptors (TLR), proinflammatory sensors of pathogens, are potential links between inflammation, infectious disease and atherosclerosis. There is increasing evidence that TLRs also recognize host-derived ligands and this also connects TLRs to diseases that may not have an etiology that is associated directly with infection. A mechanism for hyperlipidemic initiation of sterile inflammation can be postulated because oxidized lipoproteins or their component oxidized lipids have been identified as TLR ligands. Moreover, infectious agents are correlated with atherosclerosis risk. There are multiple published reports that TLR4 activation is relevant to the inflammation of atherosclerosis in mice and humans. In addition, we have identified a role for TLR2 in atherosclerosis in low density lipoprotein receptor-deficient (LDLr-/-) mice. Proatherogenic TLR2 responses to unknown endogenous or unknown endemic exogenous agonists are mediated by non-bone marrow-derived cells, which can include endothelial cells, adventitial fibroblasts and vascular smooth muscle cells. This is in contrast to the proatherogenic TLR2 response to defined synthetic exogenous agonists, which is mediated at least in part by bone marrow-derived cells, which can include lymphocytes, monocytes/macrophages, NK cells and dendritic cells. Thus, TLR2-mediated cell activation in response to endogenous and exogenous agents is proatherogenic in hyperlipidemic mice.

MD-2 binds to bacterial lipopolysaccharide.

The exact roles and abilities of the individual components of the lipopolysaccharide (LPS) receptor complex of proteins remain unclear. MD-2 is a molecule found in association with toll-like receptor 4. We produced recombinant human MD-2 to explore its LPS binding ability and role in the LPS receptor complex. MD-2 binds to highly purified rough LPS derived from Salmonella minnesota and Escherichia coli in five different assays; one assay yielded an apparent KD of 65 nm. MD-2 binding to LPS did not require LPS-binding proteins LBP and CD14; in fact LBP competed with MD-2 for LPS. MD-2 enhanced the biological activity of LPS in toll-like receptor 4-transfected Chinese hamster ovary cells but inhibited LPS activation of U373 astrocytoma cells and of monocytes in human whole blood. These data indicate that MD-2 is a genuine LPS-binding protein and strongly suggest that MD-2 could play a role in regulation of cellular activation by LPS depending on its local availability.

Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2.

The structural features of some proteins of the innate immune system involved in mediating responses to microbial pathogens are highly conserved throughout evolution. Examples include members of the Drosophila Toll (dToll) and the mammalian Toll-like receptor (TLR) protein families. Activation of Drosophila Toll is believed to occur via an endogenous peptide rather than through direct binding of microbial products to the Toll protein. In mammals there is a growing consensus that lipopolysaccharide (LPS) initiates its biological activities through a heteromeric receptor complex containing CD14, TLR4, and at least one other protein, MD-2. LPS binds directly to CD14 but whether LPS then binds to TLR4 and/or MD-2 is not known. We have used transient transfection to express human TLRs, MD-2, or CD14 alone or in different combinations in HEK 293 cells. Interactions between LPS and these proteins were studied using a chemically modified, radioiodinated LPS containing a covalently linked, UV light-activated cross-linking group ((125)I-ASD-Re595 LPS). Here we show that LPS is cross-linked specifically to TLR4 and MD-2 only when co-expressed with CD14. These data support the contention that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex.

Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism.

Leptospira interrogans are zoonotic pathogens that have been linked to a recent increased incidence of morbidity and mortality in highly populated tropical urban centers. They are unique among invasive spirochetes in that they contain outer membrane lipopolysaccharide (LPS) as well as lipoproteins. Here we show that both these leptospiral outer membrane constituents activate macrophages through CD14 and the Toll-like receptor 2 (TLR2). Conversely, it seems that TLR4, a central component for recognition of Gram-negative LPS, is not involved in cellular responses to L. interrogans. We also show that for intact L. interrogans, it is LPS, not lipoprotein, that constitutes the predominant signaling component for macrophages through a TLR2 pathway. These data provide a basis for understanding the innate immune response caused by leptospirosis and demonstrate a new ligand specificity for TLR2.

Toll-like receptor 4, but not toll-like receptor 2, is a signaling receptor for Escherichia and Salmonella lipopolysaccharides.

Two members of the mammalian Toll-like receptor (TLR) family, TLR2 and TLR4, have been implicated as receptors mediating cellular activation in response to bacterial LPS. Through the use of mAbs raised against human TLR2 and TLR4, we have conducted studies in human cell lines and whole blood to ascertain the relative contribution of these receptors to LPS induced cytokine release. We show that the contribution of TLR2 and TLR4 to LPS-induced cellular activation correlates with the relative expression levels of these two TLRs in a given cell type. In addition, we have found that significant differences in cell stimulatory activity exist between various smooth and rough LPS types that cannot be ascribed to known LPS structural features. These results suggest that impurities in the LPS may be responsible for some of the activity and this would be in agreement with recently published results of others. Upon repurification, none of the commercial LPS preparations activate cells through TLR2, but continue to stimulate cells with comparable activity through TLR4. Our results confirm recent findings that TLR4, but not TLR2, mediates cellular activation in response to LPS derived from both Escherichia coli and Salmonella minnesota. Additionally, we show that TLR4 is the predominant signaling receptor for LPS in human whole blood.

Isolation, partial characterization, and concentration in experimental sepsis of baboon lipopolysaccharide-binding protein.

Lipopolysaccharide-binding protein (LBP) is important for mediating host responses to lipopolysaccharide (LPS). The structure and properties of human, rabbit, and murine LBP have been previously described. In this study we partially characterized baboon LBP and investigated its appearance in experimental sepsis. Recurrent bacteremia was induced in baboons by infusion of live Escherichia coli organisms over a 2-hour period at 0, 24, and 48 hours. To assay baboon plasma LBP levels, an enzyme-linked immunosorbent assay with cross-reactive antibodies to human LBP was developed. Control baboon plasma LBP concentrations were 2 to 5 microg/mL. During experimental sepsis, baboon plasma LBP levels increased to between 200 and 350 microg/mL and in parallel with the increase in C-reactive protein levels. Baboon LBP was isolated from acute phase serum by ion-exchange chromatography followed by immuno-affinity chromatography. Its NH2-terminal sequence (XNPGLVARTTNKGLEYSAQE) and its molecular weight (approximately 60 kd) were determined and were proved to be highly homologous to human LBP.

Bath exposure of Atlantic halibut (Hippoglossus hippoglossus L.) yolk sac larvae to bacterial lipopolysaccharide (LPS): absorption and distribution of the LPS and effect on fish survival.

Radiolabelled bacterial lipopolysaccharide (3H-LPS) obtained from Aeromonas salmonicida subsp. salmonicida was added to the petri dishes containing yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.). The larvae were exposed either to 6.25, 12.5, 25, 50 or 100 micrograms 3H-LPS ml-1. The uptake was both dependent on the LPS concentration and the time of exposure. After 5 days of exposure, each larva contained 1.8-7.4 ng 3H-LPS dependent on the initial concentration. After 10 days of exposure each larva contained 7.0-12.4 ng LPS and after 15 days they contained 18.3-34.9 ng 3H-LPS. Fluorescence microscopic analysis of sections obtained from larvae exposed to FITC-LPS (25, 50 and 100 micrograms ml-1) for 5, 10 and 15 days, revealed fluorescence in intestinal epithelial cells, cells in the connective tissue adjacent to the intestine, in cells located between the integumental layer and yolk sac, and in some epithelial cells in the integument. By use of immunohistochemical techniques, LPS was confined to intestinal epithelial cells, lumen of excretory duct and in numerous cells in the epidermal layer. Control specimens did not contain fluorescence or were immunohistochemically negative for LPS. In groups of larvae exposed to 12.5, 25, 50 and 100 micrograms LPS ml-1, the survival was significantly increased after exposure to 50 and 100 micrograms LPS ml-1 from day 20 (96 d degree) and throughout the yolk sac period compared to untreated larvae.

Innate immune system recognition of microbial pathogens.

The long-term goal of our laboratory is to understand vertebrate host recognition of microbial pathogens. Although our work is primarily curiosity driven, it is certainly true that understanding how a host recognizes microbial pathogens should have some medical application. Probably more than 50,000 people die each year in the United States of septic shock or the systemic inflammatory response syndrome, and there is no good therapy for this problem. Understanding the molecular basis of its origin should suggest novel therapeutic approaches.

Lipopolysaccharide binding protein in acute pancreatitis.

OBJECTIVE: To assess the expression of plasma lipopolysaccharide binding protein (LBP) concentrations and its relationship to markers of the systemic inflammatory response syndrome during acute pancreatitis. DESIGN: A prospective study. SETTING: General surgical units of university teaching hospitals in the Belfast area. PATIENTS: The study included 18 patients admitted with established diagnosis of acute pancreatitis on the basis of elevated serum amylase or by contrast radiology. Patients were retrospectively stratified using the Modified Glasgow Criteria into severe (n = 7) and mild (n = 11) disease. INTERVENTIONS AND MEASUREMENTS: Blood samples were obtained at admission (day 1) and for a further 3 days for the measurement of LBP, C-reactive protein (CRP), tumor necrosis factor, and interleukin (IL)-6. Acute Physiology and Chronic Health Evaluation (APACHE) II scores were calculated on day 1 and day 2. MAIN RESULTS: LBP and CRP concentrations were significantly increased from healthy control values in acute pancreatitis patients at presentation. In the mild group LBP, CRP and IL-6 concentrations remained relatively constant throughout the study period. By comparison, severe acute pancreatitis was associated with significantly higher LBP concentrations and a marked systemic inflammatory response as evidenced by increased CRP, IL-6, and APACHE II scores. The rise in LBP occurred after the observed increase of these markers. Significant correlations were found among CRP and LBP, IL-6 and LBP, and IL-6 and APACHE II scores. There were no fatalities in the mild group, whereas four of the seven patients with severe disease died. CONCLUSIONS: LBP was significantly raised in patients with severe acute pancreatitis but would seem to be of limited use in predicting disease severity. This acute phase protein may have a role in the progression of systemic complications associated with acute pancreatitis.

Structure-function analysis of CD14 as a soluble receptor for lipopolysaccharide.

CD14 is a glycophosphatidylinositol-linked protein expressed by myeloid cells and also circulates as a plasma protein lacking the glycophosphatidylinositol anchor. Both membrane and soluble CD14 function to enhance activation of cells by lipopolysaccharide (LPS), which we refer to as receptor function. We have previously reported the LPS binding and cell activation functions of a group of five deletion mutants of CD14 (Viriyakosol, S., and Kirkland, T.N. (1995) J. Biol. Chem. 270, 361-368). We have now studied the functional impact of these mutations on soluble CD14. We found that some deletions that abrogated LPS binding in membrane CD14 have no effect on LPS binding in soluble CD14. In fact, some of the soluble CD14 deletion mutants bound LPS with an apparent higher affinity than wild-type CD14. Furthermore, we found that all five deletions essentially ablated soluble CD14 LPS receptor function, whereas only two of the deletions completely destroyed membrane CD14 LPS receptor function. Some of the mutants were able to compete with wild-type CD14 in soluble CD14-dependent assays of cellular activation. We concluded that the soluble and membrane forms of CD14 have different structural determinants for LPS receptor function.

Membrane-anchored forms of lipopolysaccharide (LPS)-binding protein do not mediate cellular responses to LPS independently of CD14.

Inflammatory responses of myeloid cells to LPS are mediated through CD14, a glycosylphosphatidylinositol-anchored receptor that binds LPS. Since CD14 does not traverse the plasma membrane and alternatively anchored forms of CD14 still enable LPS-induced cellular activation, the precise role of CD14 in mediating these responses remains unknown. To address this, we created a transmembrane and a glycosylphosphatidylinositol-anchored form of LPS-binding protein (LBP), a component of serum that binds and transfers LPS to other molecules. Stably transfected Chinese hamster ovary (CHO) fibroblast and U373 astrocytoma cell lines expressing membrane-anchored LBP (mLBP), as well as separate CHO and U373 cell lines expressing membrane CD14 (mCD14), were subsequently generated. Under serum-free conditions, CHO and U373 cells expressing mCD14 responded to as little as 0.1 ng/ml of LPS, as measured by NF-kappaB activation as well as ICAM and IL-6 production. Conversely, the vector control and mLBP-expressing cell lines did not respond under serum-free conditions even in the presence of more than 100 ng/ml of LPS. All the cell lines exhibited responses to less than 1 ng/ml of LPS in the presence of the soluble form of CD14, demonstrating that they are still capable of LPS-induced activation. Taken together, these results demonstrate that mLBP, a protein that brings LPS to the cell surface, does not mediate cellular responses to LPS independently of CD14. These findings suggest that CD14 performs a more specific role in mediating responses to LPS than that of simply bringing LPS to the cell surface.

Endotoxin interactions with lipopolysaccharide-responsive cells.

Recent work has identified two proteins that work together to enable many cell types to respond to endotoxin. These two proteins, lipopolysaccharide (LPS) binding protein (LBP) and CD14, also participate in cellular internalization of endotoxin, which may occur independently of cellular activation. Current work with antibodies to LBP and CD14 as well as "knockout" mice in the context of LPS-initiated endotoxic shock suggests that inhibition of this pathway could be therapeutically useful. These observations point to the need to identify new molecules that mediate LPS-initiated transmembrane signaling and internalization of LPS-protein complexes.

Recognition of gram-negative bacteria and endotoxin by the innate immune system.

Until about 10 years ago the exact mechanisms controlling cellular responses to the endotoxin - or lipopolysaccharide (LPS) - of Gram-negative bacteria were unknown. Now a considerable body of evidence supports a model where LPS or LPS-containing particles (including intact bacteria) form complexes with a serum protein known as LPS-binding protein; the LPS in this complex is subsequently transferred to another protein which binds LPS, CD14. The latter is found on the plasma membrane of most cell types of the myeloid lineage as well as in the serum in its soluble form; LPS binding to these two forms of CD14 results in the activation of cell types of myeloid and nonmyeloid lineages, respectively.

Binding of bacterial peptidoglycan to CD14.

The hypothesis that soluble peptidoglycan (sPGN, a macrophage-activator from Gram-positive bacteria) binds to CD14 (a lipopolysaccharide (LPS) receptor) was tested. sPGN specifically bound to CD14 in the following three assays: binding of soluble 32P-CD14 (sCD14) to agarose-immobilized sPGN, enzyme-linked immunosorbent assay, and photoaffinity cross-linking. sCD14 also specifically bound to agarose-immobilized muramyl dipeptide or GlcNAc-muramyl dipeptide but not to PGN pentapeptide. Binding of sCD14 to both sPGN and ReLPS (where ReLPS is LPS from Salmonella minnesota Re 595) was competitively inhibited by unlabeled sCD14, 1-152 N-terminal fragment of sCD14, sPGN, smooth LPS, ReLPS, lipid A, and lipoteichoic acid but not by dextran, dextran sulfate, heparin, ribitol teichoic acid, or soluble low molecular weight PGN fragments. Binding of sCD14 to sPGN was slower than to ReLPS but of higher affinity (KD = 25 nM versus 41 nM). LPS-binding protein (LBP) increased the binding of sCD14 to sPGN by adding another lower affinity KD and another higher Bmax, but for ReLPS, LBP increased the affinity of binding by yielding two KD with significantly higher affinity (7.1 and 27 nM). LBP also enhanced inhibition of sCD14 binding by LPS, ReLPS, and lipid A. Binding of sCD14 to both sPGN and ReLPS was inhibited by anti-CD14 MEM-18 mAb, but other anti-CD14 mAbs showed differential inhibition, suggesting conformational binding sites on CD14 for sPGN and LPS, that are partially identical and partially different.

Roles for LBP and soluble CD14 in cellular uptake of LPS.

Roles for LBP and CD14 in the LPS dependent activation of a wide variety of cells have been established. In the work described here, we describe roles for these proteins in the binding and uptake of LPS by cells which express membrane CD14 and those which do not. Surprisingly, cell activation and LPS uptake appear to be independent phenomena with different protein requirements.

Cell activation mediated by glycosylphosphatidylinositol-anchored or transmembrane forms of CD14.

CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein which functions as a receptor on myeloid cells for ligands derived from microbial pathogens such as lipopolysaccharide (LPS). We have studied the importance of the GPI tail of CD14 in signalling with the promonocytic cell line THP-1 expressing recombinant CD14 in a GPI-anchored form (THP1-wtCD14 cells) or in a transmembrane form (THP1-tmCD14). We found that, like other GPI-anchored molecules, GPI-anchored CD14 was recovered mainly from a Triton X-100-insoluble fraction, whereas transmembrane CD14 was fully soluble in Triton X-100. LPS induced cell activation of THP1-wtCD14 and of THP1-tmCD14 (protein tyrosine kinase phosphorylation, NF-kappaB activation, and cytokine production) in a very similar manner. However, anti-CD14 antibody-induced cross-linking caused a rapid calcium mobilization signal only in GPI-anchored CD14 cells. Studies with pharmacologic inhibitors of intracellular signalling events implicate phospholipase C and protein tyrosine kinases in the genesis of this antibody-induced calcium signal. Our results suggest that GPI anchoring and CD14 targeting to glycolipid-rich membrane microdomains are not required for LPS-mediated myeloid cell activation. GPI anchoring may however be important for other signalling functions, such as those events reflected by antibody cross-linking.

Lactoferrin inhibits the endotoxin interaction with CD14 by competition with the lipopolysaccharide-binding protein.

Human lactoferrin (hLf), a glycoprotein released from neutrophil granules during inflammation, and the lipopolysaccharide (LPS)-binding protein (LBP), an acute-phase serum protein, are known to bind to the lipid A of LPS. The LPS-binding sites are located in the N-terminal regions of both proteins, at amino acid residues 28 to 34 of hLf and 91 to 108 of LBP. Both of these proteins modulate endotoxin activities, but they possess biologically antagonistic properties. In this study, we have investigated the competition between hLf and recombinant human LBP (rhLBP) for the binding of Escherichia coli 055:B5 LPS to the differentiated monocytic THP-1 cell line. Our studies revealed that hLf prevented the rhLBP-mediated binding of LPS to the CD14 receptor on cells. Maximal inhibition of LPS-cell interactions by hLf was raised when both hLf and rhLBP were simultaneously added to LPS or when hLf and LPS were mixed with cells 30 min prior to the incubation with rhLBP. However, when hLf was added 30 min after the interaction of rhLBP with LPS, the binding of the rhLPS-LBP complex to CD14 could not be reversed. These observations indicate that hLf competes with rhLBP for the LPS binding and therefore interferes with the interaction of LPS with CD14. Furthermore, experiments involving competitive binding of the rhLBP-LPS complex to cells with two recombinant mutated hLfs show that in addition to residues 28 to 34, another basic cluster which contains residues 1 to 5 of hLf competes for the binding to LPS. Basic sequences homologous to residues 28 to 34 of hLf were evidenced on LPS-binding proteins such as LBP, bactericidal/permeability-increasing protein, and Limulus anti-LPS factor.

Phagocytosis of gram-negative bacteria by a unique CD14-dependent mechanism.

THP-1-derived cell lines were stably transfected with constructs encoding glycophosphatidylinositol (GPI)-anchored or transmembrane forms of human CD14. CD14 expression was associated with enhanced phagocytosis of serum (heat-inactivated)-opsonized Escherichia coli (opEc). Both the GPI-anchored and transmembrane forms of CD14 supported phagocytosis of opEc equally well. Lipopolysaccharide-binding protein (LBP) played a role in CD14-dependent phagocytosis as evidenced by inhibition of CD14-dependent phagocytosis of opEc with anti-LBP monoclonal antibody (mAb) and by enhanced phagocytosis of E. coli opsonized with purified LBP. CD14-dependent phagocytosis was inhibited by a phosphatidylinositol (PI) 3-kinase inhibitor (wortmannin) and a protein tyrosine kinase inhibitor (tyrphostin 23) but not a protein kinase C inhibitor (bisindolyl-maleimide) or a divalent cation chelator (ethylenediaminetetraacetate). Anti-LBP mAb 18G4 and anti-CD14 mAb 18E12 were used to differentiate between the pathways involved in CD14-dependent phagocytosis and CD14-dependent cell activation. F(ab')2 fragments of 18G4, a mAb to LBP that does not block cell activation, inhibited ingestion of opEc by THP1-wtCD14 cells. 18E12 (an anti-CD14 mAb that does not block LPS binding to CD14 but does inhibit CD14-dependent cell activation) did not inhibit phagocytosis of LBP-opEc by THP1-wtCD14 cells. Furthermore, CD14-dependent phagocytosis was not inhibited by anti-CD18 (CR3 and CR4 beta-chain) or anti-Fcgamma receptor mAb.