Peter Tobias | Scripps Research Institute (original) (raw)
Papers by Peter Tobias
Innate immunity, 2012
Signaling through MyD88, an adaptor utilized by all TLRs except TLR3, is pro-atherogenic; however... more Signaling through MyD88, an adaptor utilized by all TLRs except TLR3, is pro-atherogenic; however, it is unknown whether signaling through TIR-domain-containing adaptor-inducing interferon-β (TRIF), an adaptor used only by TLRs 3 and 4, is relevant to atherosclerosis. We determined that the TRIF(Lps2) lack-of-function mutation was atheroprotective in hyperlipidemic low density lipoprotein (LDL) receptor knockout (LDLr(-/-)) mice. LDLr(-/-) mice were crossed with either TRIF(Lps2) or TLR3 knockout mice. After feeding an atherogenic diet for 10-15 wks, atherosclerotic lesions in the heart sinus and aorta were quantitated. LDLr(-/-) mice with TRIF(Lps2) were significantly protected from atherosclerosis. TRIF(Lps2) led to a reduction in cytokines secreted from peritoneal macrophages (Mϕ) in response to hyperlipidemia. Moreover, heart sinus valves from hyperlipidemic LDLr(-/-)TRIF(Lps2) mice had significantly fewer lesional Mϕ. However, LDLr(-/-) mice deficient in TLR3 showed some enhanc...
The Journal of clinical investigation, 2005
Epidemiologic evidence has established a relationship between microbial infection and atheroscler... more Epidemiologic evidence has established a relationship between microbial infection and atherosclerosis. Mammalian TLRs provide clues on the mechanism of this inflammatory cascade. TLR2 has a large ligand repertoire that includes bacterial-derived exogenous and possibly host-derived endogenous ligands. In atherosclerosis-susceptible low-density lipoprotein receptor-deficient (Ldlr-/-) mice, complete deficiency of TLR2 led to a reduction in atherosclerosis. However, with BM transplantation, loss of TLR2 expression from BM-derived cells had no effect on disease progression. This suggested that an unknown endogenous TLR2 agonist influenced lesion progression by activating TLR2 in cells that were not of BM cell origin. Moreover, with intraperitoneal administration of a synthetic TLR2/TLR1 agonist, Pam3CSK4, disease burden was dramatically increased in Ldlr-/- mice. A complete deficiency of TLR2 in Ldlr-/- mice, as well as a deficiency of TLR2 only in BM-derived cells in Ldlr-/- mice, led ...
Journal of Lipid Research, 2008
Atherosclerosis is inflammation of the vessel wall of the arterial tree. This inflammation arises... more Atherosclerosis is inflammation of the vessel wall of the arterial tree. This inflammation arises at specific areas that experience disturbed blood flow such as bifurcations and the lesser curvature of the aortic arch. Although all endothelial cells are exposed to comparable levels of circulating plasma cholesterol, only endothelial cells overlaying lesions display an inflamed phenotype. This occurs even in the absence of any additional exacerbating disease factors because blood flow controls the expression of Toll-like receptors (TLR), which are initiators of cellular activation and inflammation. TLR2-and 4-expression exert an overall proatherogenic effect in hyperlipidemic mice. TLR activation of the endothelium promotes lipid accumulation and leukocyte accumulation within lesions.-Curtiss, L. K., and P. S. Tobias. Emerging role of Toll-like receptors in atherosclerosis.
Journal of Lipid Research, 2012
Biochemical Society Transactions, 2007
At one time, atherosclerosis was thought to be a simple lipid storage disease. However, it is now... more 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 hav...
The Journal of Immunology, 2007
TLRs are an evolutionarily conserved family of cell membrane proteins believed to play a signific... more TLRs are an evolutionarily conserved family of cell membrane proteins believed to play a significant role in innate immunity and the response to tissue injury, including that induced by ischemia. TLR signaling pathways activate transcription factors that regulate expression of prosurvival proteins, as well as proinflammatory cytokines and chemokines through one of two proximal adapter proteins, MyD88 or Toll/IL-1R domain-containing adaptor-inducing IFN-β (Trif). Our study defines the constitutive protein expression of TLR2 in kidneys of humans and mice, and provides insight into the signaling mechanisms by which a deficiency of TLR2 protects from ischemic organ injury. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in TLR2, MyD88, Trif, and MyD88 × Trif. TLR2 protein was evident in many cell types in the kidney, including renal tubules of the outer stripe of the medulla, glomeruli, and in the renal vasculature. The pattern of pro...
Immunologic Research, 2006
Arteriosclerosis, Thrombosis, and Vascular Biology, 2011
Objective—The purpose of this study was to determine whether myeloid differentiation factor 88 (M... more Objective—The purpose of this study was to determine whether myeloid differentiation factor 88 (MyD88) and its related Toll-like receptors (TLRs) 2 and 4 contributed to the development of angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) and atherosclerosis.Methods and Results—AngII was infused into either apoE−/−or LDL receptor (LDLR)−/−male mice that were either MyD88+/+or−/−. MyD88 deficiency profoundly reduced AngII-induced AAAs and atherosclerosis in both strains. To define whether deficiency of specific TLRs had similar effects, AngII was infused into LDLR−/−mice that were also deficient in either TLR2 or TLR4. TLR2 deficiency had no effect on AAA development but inhibited atherosclerosis. In contrast, TLR4 deficiency attenuated both AAAs and atherosclerosis. To resolve whether MyD88 and TLR4 exerted their effects through cells of hematopoietic lineage, LDLR−/−mice were lethally irradiated and repopulated with bone marrow-derived cells from either MyD88 or TLR4 ...
Journal of Leukocyte Biology, 1990
Human peripheral blood neutrophils are primed, or enabled to respond to formyl peptide. by prior ... more Human peripheral blood neutrophils are primed, or enabled to respond to formyl peptide. by prior exposure to bacterial lipopolysaccharide (LPS). The activity of LPS and the size of its aggregates are altered by plasma constituents such as high density lipoprotein (HDL) and the recently discovered acute phase reactant lipopolysaccharide binding protein (LBP) Tobias et al.: J. Exp. Med. 164,777, 1986]. The ability of LPS, LPS–LBP, and LPS–HDL complexes to activate a number of cellular responses have been compared. LPS–LBP and LPS–HDL were prepared using LBP and HDL from rabbit serum. LPS from Salmonella minnesota Re595 and its LPS–LBP and LPS–HDL complexes differed in their ability to prime PMN O2− production in response to formyl peptide (f-Nle-Leu-Phe-Nle-Tyr-Leu [FNLPNTL]). Human PMN prepared under conditions in which O2 production is minimal (<1 nmol O2−/106 PMN/10 min) after exposure to 10-7 M FNLPNTL can be primed with 0.1–100 ng/ml LPS in a dose- and time-dependent manner to...
Journal of Clinical Investigation, 1994
Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effe... more Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effect of Escherichia coli 011 1:B4 LPS on movement of '4C-BSA across bovine pulmonary artery EC monolayers. In the presence of serum, a 6-h LPS exposure augmented (P < 0.001) transendothelial 14C-BSA flux compared with the media control at concentrations 2 0.5 ng/ml, and LPS (10 ng/ml) exposures of 2 2-h increased (P < 0.005) the flux. In the absence of serum, LPS concentrations of up to 10 Ag/ml failed to increase 14C-BSA flux at 6 h. The addition of 10% serum increased EC sensitivity to the LPS stimulus by > 10,000-fold. LPS (10 ng/ml, 6 h) failed to increase 14C-BSA flux at serum concentrations < 0.5%, and maximum LPS-induced increments could be generated in the presence of. 2.5%. LPS-binding protein (LBP) and soluble CD14 (sCD14) could each satisfy this serum requirement; either anti-LBP or anti-CD14 antibody each totally blocked (P < 0.00005) the LPS-induced changes in endothelial barrier function. LPS-LBP had a more rapid onset than did LPS-sCD14. The LPS effect in the presence of both LBP and sCD14 exceeded the effect in the presence of either protein alone. These data suggest that LBP and sCD14 each independently functions as an accessory molecule for LPS presentation to the non-CD14-bearing endothelial surface. However, in the presence of serum both molecules are required.
Journal of Biological Chemistry, 1997
Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (... more Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are closely related LPS-binding proteins whose binding to LPS has markedly different functional consequences. To gain better insight into the possible basis of these functional differences, the physical properties of LBP-LPS and BPI-LPS complexes have been compared in this study by sedimentation, light scattering, and fluorescence analyses. These studies reveal dramatic differences in the physical properties of LPS complexed to LBP versus BPI. They suggest that of the two proteins, only LBP can disperse LPS aggegates. However, BPI can enhance both the sedimentation velocity and apparent size of LPS aggregates while inhibiting LPS-LBP binding even at very low (1:40 to 1:20) BPI:LPS molar ratios. The lipopolysaccharide (LPS)-binding protein 1 (LBP) and the bactericidal/permeability-increasing protein (BPI) are both LPS-interactive mammalian proteins with approximately 45% amino acid sequence identity (1, 2). LPS is considered to be the principal component of Gram-negative bacteria that alerts the host to invading bacteria and triggers defensive responses (3, 4). These responses are usually beneficial and effective but may also become excessive and lead to endotoxic shock (3-5). Both LBP and BPI modulate the bioactivity of LPS (2, 3, 5). LBP is a plasma protein that catalyzes the transfer of LPS from LPS aggregates to other LPS-binding proteins (3, 6-9). Prominent among these is CD14, a surface molecule of myeloid cells that is also present in the circulation as a soluble protein. LBP and CD14 together represent the main pathway by which cells recognize low concentrations of LPS and are stimulated to respond to Gram-negative bacteria (3, 10, 11). In contrast to the LPS-stimulatory properties of LBP, binding of LPS by BPI results in inhibition of the bioactivities of LPS (2). BPI is produced by polymorphonuclear leukocytes and stored in its azurophilic granules (12, 13). It contributes substantially to both the intracellular and extracellular antibacterial activity of polymorphonuclear leukocyte-rich inflammatory exudates toward Gram-negative bacteria (14, 15). The high affinity of BPI
Journal of Biological Chemistry, 2003
Lipopolysaccharide (LPS)-binding protein (LBP) is an acute phase reactant that may play a dual ro... more Lipopolysaccharide (LPS)-binding protein (LBP) is an acute phase reactant that may play a dual role in vivo, both potentiating and decreasing cell responses to bacterial LPS. Whereas low concentrations of LBP potentiate cell stimulation by transferring LPS to CD14, high LBP concentrations inhibit cell responses to LPS. One inhibitory mechanism involves the ability of LBP to neutralize LPS by transferring it to plasma lipoproteins, whereas other inhibitory mechanisms, such as the one described here, do not require exogenous lipoproteins. Here we show that LBP can inhibit monocyte responses to LPS that has already bound to membrane-bound CD14 (mCD14) on the cell surface. LBP caused rapid dissociation of LPS from mCD14 as measured by the ability of LBP to inhibit cross-linking of a radioiodinated, photoactivatable LPS derivative to mCD14. Whereas LBP removed up to 75% of the mCD14-bound LPS in 10 min, this was not accompanied by extensive release of the LPS from the cells. The cross-linking data suggest that much of the LPS that remained bound to the cells was associated with LBP. The ability of LBP to inhibit cell responses could not be explained by its effect on LPS internalization, because LBP did not significantly increase the internalization of the cell-bound LPS. In cell-free LPS cross-linking experiments, LBP inhibited the transfer of LPS from soluble CD14 to soluble MD-2. Our data support the hypothesis that LBP can inhibit cell responses to LPS by inhibiting LPS transfer from mCD14 to the Toll-like receptor 4-MD-2 signaling receptor. Lipopolysaccharide (LPS 1 ; endotoxin), an abundant component of the outer membrane of Gram-negative bacteria, is one
Journal of Biological Chemistry, 1998
The hypothesis that soluble peptidoglycan (sPGN, a macrophage-activator from Gram-positive bacter... more 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 32 P-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 (K D ؍ 25 nM versus 41 nM). LPS-binding protein (LBP) increased the binding of sCD14 to sPGN by adding another lower affinity K D and another higher B max , but for ReLPS, LBP increased the affinity of binding by yielding two K D 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. Peptidoglycan (PGN) 1 is a polymer of alternating GlcNAc and MurNAc cross-linked by short peptides, present in the cell walls of all bacteria (1). PGN, similar to lipopolysaccharide (LPS) from the cell walls of Gram-negative bacteria, can reproduce most of the clinical manifestations of bacterial infections, including fever, acute-phase response,
Journal of Biological Chemistry, 1997
The stimulation of nonmyeloid cells by lipopolysaccharide (LPS) is mediated by the serum protein,... more The stimulation of nonmyeloid cells by lipopolysaccharide (LPS) is mediated by the serum protein, soluble CD14 (sCD14). We have examined the interaction of sCD14 with whole cells using a biologically active radiolabeled sCD14 molecule as a ligand. Specific binding of sCD14 to nonmyeloid cells is detected only when it is first incubated with both LPS and the serum LPS-binding protein (LBP). Through the use of an anti-CD14 monoclonal antibody, we demonstrate that sCD14 must interact with LPS in order for cellular binding to occur. Although LBP is traditionally known to function as a catalyst in the transfer of LPS to sCD14, our results reveal that LBP is actually a physical part of sCD14containing,cell-associatingcomplexes.TheLPS-andLBPdependent cell surface binding of sCD14 appears to be distinct from events leading to cell stimulation, since certain anti-CD14 and anti-LBP monoclonal antibodies have different effects on cellular binding versus cellular activation. Bound sCD14 is internalized, indicating that the LBP-and LPS-dependent binding of sCD14 may represent a novel general mechanism by which nonmyeloid cells clear LPS.
Journal of Biological Chemistry, 2001
Journal of Biological Chemistry, 2006
Toll-like receptor 4 and MD-2 form a receptor for lipopolysaccharide (LPS), a major constituent o... more Toll-like receptor 4 and MD-2 form a receptor for lipopolysaccharide (LPS), a major constituent of Gram-negative bacteria. MD-2 is a 20-25-kDa extracellular glycoprotein that binds to Tolllike receptor 4 (TLR4) and LPS and is a critical part of the LPS receptor. Here we have shown that the level of MD-2 expression regulates TLR4 activation by LPS. Using site-directed mutagenesis, we have found that glycosylation has no effect on MD-2 function as a membrane receptor for LPS. We used alanine-scanning mutagenesis to identify regions of human MD-2 that are important for TLR4 and LPS binding. We found that mutation in the N-terminal 46 amino acids of MD-2 did not substantially diminish LPS activation of Chinese hamster ovary (CHO) cells co-transfected with TLR4 and mutant MD-2. The residues 46-50 were important for LPS activation but not LPS binding. The residues 79-83, 121-124, and 125-129 are identified as important in LPS activation but not surface expression of membrane MD-2. The function of soluble MD-2 is somewhat more sensitive to mutation than membrane MD-2. Our results suggest that the 46-50 and 127-131 regions of soluble MD-2 bind to TLR4. The region 79-120 is not involved in LPS binding but affects monomerization of soluble MD-2 as well as TLR4 binding. We define the LPS binding region of monomeric soluble MD-2 as a cluster of basic residues 125-131. Studies on both membrane and soluble MD-2 suggest that domains of MD-2 for TLR4 and LPS binding are separate as well as overlapping. By mapping these regions on a three-dimensional model, we show the likely binding regions of MD-2 to TLR4 and LPS.
Immunity, 2006
CD14 is a well-known pattern-recognition receptor in the innate immune system. Here, we show that... more CD14 is a well-known pattern-recognition receptor in the innate immune system. Here, we show that CD14 enhances double-stranded RNA (dsRNA)-mediated Toll-like receptor 3 (TLR3) activation. Bone marrowderived macrophages (BMDMs) from CD14 2/2 mice exhibited impaired responses to polyinosine-polycytidylic acid (pIpC) and reduced production of inflammatory cytokines. CD14 2/2 mice injected with pIpC also showed impaired cytokine production. When tested with [ 32 P] labeled pIpC small fragments (pIpCsf) that maintain the inflammatory activity of crude pIpC, CD14 directly bound pIpCsf and mediated cellular uptake of pIpCsf. Our data show that TLR3 is intracellular and directly interacts with CD14. Internalized pIpCsf was localized in the lysosomes via the endosomes. In unstimulated cells, neither CD14 nor TLR3 was detected in the lysosomes. However, TLR3 was localized in the lysosomes as was CD14 once the cells took up pIpC. We also observed that internalized pIpCsf colocalized with CD14 and TLR3. Consequently, CD14 mediates pIpC uptake and enhances TLR3 signaling.
Clinical Infectious Diseases, 1999
Recent work has identified two proteins that work together to enable many cell types to respond t... more 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 LPSinitiated transmembrane signaling and internalization of LPS-protein complexes. This article is part of a series of papers presented at a symposium entitled from rabbit blood inhibited responses to LPS [21], that LBP ''Molecular Mechanisms of Microbial Host Cell Interactions in Periodontal enhanced the LPS-dependent activation of rabbit peritoneal Disease'' that was held on 14
Circulation Research, 2004
Innate immune system activation is associated with atherosclerotic lesion development. The specif... more Innate immune system activation is associated with atherosclerotic lesion development. The specific sites of lesion development are believed to be defined by the shear stress of blood flow. Consequently, we investigated the responsiveness of human coronary artery endothelial cells (HCAECs) to Toll-like receptor (TLR) 2 and 4 agonists in an in vitro model of chronic laminar flow. HCAECs under chronic laminar flow were found to be normally responsive to lipopolysaccharide (and tumor necrosis factor) in terms of E-selectin expression but were found to be hyporesponsive to stimulation with the specific TLR2 ligands macrophage activating lipopeptide-2, PAM 2 -Cys, and Lip19; this was observed to be attributable to downregulation of TLR2 transcription and protein expression. We found that laminar flow induced SP1 serine phosphorylation by protein kinase CK2 and thereby blocked SP1 binding to the TLR2 promoter, which is required for TLR2 expression. This regulatory mechanism also blocked l...
Biochemical Journal, 2005
SP-A (surfactant protein A) is a lipid-binding collectin primarily involved in innate lung immuni... more SP-A (surfactant protein A) is a lipid-binding collectin primarily involved in innate lung immunity. SP-A interacts with the bacterial rough LPS (lipopolysaccharide) Re-LPS (Re595 mutant of LPS from Salmonella minnesota), but not with smooth LPS. In the present study, we first examined the characteristics of the interaction of human SP-A with Re-LPS. Fluorescence intensity and anisotropy measurements of FITC-labelled Re-LPS in the presence and absence of SP-A indicated that SP-A bound to Re-LPS in solution in a Ca2+-independent manner, with a dissociation constant of 2.8×10−8 M. In the presence of calcium, a high-mobility complex of SP-A and [3H]Rb-LPS (Rb mutant of LPS from Escherichia coli strain LCD 25) micelles was formed, as detected by sucrose density gradients. Re-LPS aggregation induced by SP-A was further characterized by light scattering. On the other hand, human SP-A inhibited TNF-α (tumour necrosis factor-α) secretion by human macrophage-like U937 cells stimulated with e...
Innate immunity, 2012
Signaling through MyD88, an adaptor utilized by all TLRs except TLR3, is pro-atherogenic; however... more Signaling through MyD88, an adaptor utilized by all TLRs except TLR3, is pro-atherogenic; however, it is unknown whether signaling through TIR-domain-containing adaptor-inducing interferon-β (TRIF), an adaptor used only by TLRs 3 and 4, is relevant to atherosclerosis. We determined that the TRIF(Lps2) lack-of-function mutation was atheroprotective in hyperlipidemic low density lipoprotein (LDL) receptor knockout (LDLr(-/-)) mice. LDLr(-/-) mice were crossed with either TRIF(Lps2) or TLR3 knockout mice. After feeding an atherogenic diet for 10-15 wks, atherosclerotic lesions in the heart sinus and aorta were quantitated. LDLr(-/-) mice with TRIF(Lps2) were significantly protected from atherosclerosis. TRIF(Lps2) led to a reduction in cytokines secreted from peritoneal macrophages (Mϕ) in response to hyperlipidemia. Moreover, heart sinus valves from hyperlipidemic LDLr(-/-)TRIF(Lps2) mice had significantly fewer lesional Mϕ. However, LDLr(-/-) mice deficient in TLR3 showed some enhanc...
The Journal of clinical investigation, 2005
Epidemiologic evidence has established a relationship between microbial infection and atheroscler... more Epidemiologic evidence has established a relationship between microbial infection and atherosclerosis. Mammalian TLRs provide clues on the mechanism of this inflammatory cascade. TLR2 has a large ligand repertoire that includes bacterial-derived exogenous and possibly host-derived endogenous ligands. In atherosclerosis-susceptible low-density lipoprotein receptor-deficient (Ldlr-/-) mice, complete deficiency of TLR2 led to a reduction in atherosclerosis. However, with BM transplantation, loss of TLR2 expression from BM-derived cells had no effect on disease progression. This suggested that an unknown endogenous TLR2 agonist influenced lesion progression by activating TLR2 in cells that were not of BM cell origin. Moreover, with intraperitoneal administration of a synthetic TLR2/TLR1 agonist, Pam3CSK4, disease burden was dramatically increased in Ldlr-/- mice. A complete deficiency of TLR2 in Ldlr-/- mice, as well as a deficiency of TLR2 only in BM-derived cells in Ldlr-/- mice, led ...
Journal of Lipid Research, 2008
Atherosclerosis is inflammation of the vessel wall of the arterial tree. This inflammation arises... more Atherosclerosis is inflammation of the vessel wall of the arterial tree. This inflammation arises at specific areas that experience disturbed blood flow such as bifurcations and the lesser curvature of the aortic arch. Although all endothelial cells are exposed to comparable levels of circulating plasma cholesterol, only endothelial cells overlaying lesions display an inflamed phenotype. This occurs even in the absence of any additional exacerbating disease factors because blood flow controls the expression of Toll-like receptors (TLR), which are initiators of cellular activation and inflammation. TLR2-and 4-expression exert an overall proatherogenic effect in hyperlipidemic mice. TLR activation of the endothelium promotes lipid accumulation and leukocyte accumulation within lesions.-Curtiss, L. K., and P. S. Tobias. Emerging role of Toll-like receptors in atherosclerosis.
Journal of Lipid Research, 2012
Biochemical Society Transactions, 2007
At one time, atherosclerosis was thought to be a simple lipid storage disease. However, it is now... more 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 hav...
The Journal of Immunology, 2007
TLRs are an evolutionarily conserved family of cell membrane proteins believed to play a signific... more TLRs are an evolutionarily conserved family of cell membrane proteins believed to play a significant role in innate immunity and the response to tissue injury, including that induced by ischemia. TLR signaling pathways activate transcription factors that regulate expression of prosurvival proteins, as well as proinflammatory cytokines and chemokines through one of two proximal adapter proteins, MyD88 or Toll/IL-1R domain-containing adaptor-inducing IFN-β (Trif). Our study defines the constitutive protein expression of TLR2 in kidneys of humans and mice, and provides insight into the signaling mechanisms by which a deficiency of TLR2 protects from ischemic organ injury. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in TLR2, MyD88, Trif, and MyD88 × Trif. TLR2 protein was evident in many cell types in the kidney, including renal tubules of the outer stripe of the medulla, glomeruli, and in the renal vasculature. The pattern of pro...
Immunologic Research, 2006
Arteriosclerosis, Thrombosis, and Vascular Biology, 2011
Objective—The purpose of this study was to determine whether myeloid differentiation factor 88 (M... more Objective—The purpose of this study was to determine whether myeloid differentiation factor 88 (MyD88) and its related Toll-like receptors (TLRs) 2 and 4 contributed to the development of angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) and atherosclerosis.Methods and Results—AngII was infused into either apoE−/−or LDL receptor (LDLR)−/−male mice that were either MyD88+/+or−/−. MyD88 deficiency profoundly reduced AngII-induced AAAs and atherosclerosis in both strains. To define whether deficiency of specific TLRs had similar effects, AngII was infused into LDLR−/−mice that were also deficient in either TLR2 or TLR4. TLR2 deficiency had no effect on AAA development but inhibited atherosclerosis. In contrast, TLR4 deficiency attenuated both AAAs and atherosclerosis. To resolve whether MyD88 and TLR4 exerted their effects through cells of hematopoietic lineage, LDLR−/−mice were lethally irradiated and repopulated with bone marrow-derived cells from either MyD88 or TLR4 ...
Journal of Leukocyte Biology, 1990
Human peripheral blood neutrophils are primed, or enabled to respond to formyl peptide. by prior ... more Human peripheral blood neutrophils are primed, or enabled to respond to formyl peptide. by prior exposure to bacterial lipopolysaccharide (LPS). The activity of LPS and the size of its aggregates are altered by plasma constituents such as high density lipoprotein (HDL) and the recently discovered acute phase reactant lipopolysaccharide binding protein (LBP) Tobias et al.: J. Exp. Med. 164,777, 1986]. The ability of LPS, LPS–LBP, and LPS–HDL complexes to activate a number of cellular responses have been compared. LPS–LBP and LPS–HDL were prepared using LBP and HDL from rabbit serum. LPS from Salmonella minnesota Re595 and its LPS–LBP and LPS–HDL complexes differed in their ability to prime PMN O2− production in response to formyl peptide (f-Nle-Leu-Phe-Nle-Tyr-Leu [FNLPNTL]). Human PMN prepared under conditions in which O2 production is minimal (<1 nmol O2−/106 PMN/10 min) after exposure to 10-7 M FNLPNTL can be primed with 0.1–100 ng/ml LPS in a dose- and time-dependent manner to...
Journal of Clinical Investigation, 1994
Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effe... more Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effect of Escherichia coli 011 1:B4 LPS on movement of '4C-BSA across bovine pulmonary artery EC monolayers. In the presence of serum, a 6-h LPS exposure augmented (P < 0.001) transendothelial 14C-BSA flux compared with the media control at concentrations 2 0.5 ng/ml, and LPS (10 ng/ml) exposures of 2 2-h increased (P < 0.005) the flux. In the absence of serum, LPS concentrations of up to 10 Ag/ml failed to increase 14C-BSA flux at 6 h. The addition of 10% serum increased EC sensitivity to the LPS stimulus by > 10,000-fold. LPS (10 ng/ml, 6 h) failed to increase 14C-BSA flux at serum concentrations < 0.5%, and maximum LPS-induced increments could be generated in the presence of. 2.5%. LPS-binding protein (LBP) and soluble CD14 (sCD14) could each satisfy this serum requirement; either anti-LBP or anti-CD14 antibody each totally blocked (P < 0.00005) the LPS-induced changes in endothelial barrier function. LPS-LBP had a more rapid onset than did LPS-sCD14. The LPS effect in the presence of both LBP and sCD14 exceeded the effect in the presence of either protein alone. These data suggest that LBP and sCD14 each independently functions as an accessory molecule for LPS presentation to the non-CD14-bearing endothelial surface. However, in the presence of serum both molecules are required.
Journal of Biological Chemistry, 1997
Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (... more Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are closely related LPS-binding proteins whose binding to LPS has markedly different functional consequences. To gain better insight into the possible basis of these functional differences, the physical properties of LBP-LPS and BPI-LPS complexes have been compared in this study by sedimentation, light scattering, and fluorescence analyses. These studies reveal dramatic differences in the physical properties of LPS complexed to LBP versus BPI. They suggest that of the two proteins, only LBP can disperse LPS aggegates. However, BPI can enhance both the sedimentation velocity and apparent size of LPS aggregates while inhibiting LPS-LBP binding even at very low (1:40 to 1:20) BPI:LPS molar ratios. The lipopolysaccharide (LPS)-binding protein 1 (LBP) and the bactericidal/permeability-increasing protein (BPI) are both LPS-interactive mammalian proteins with approximately 45% amino acid sequence identity (1, 2). LPS is considered to be the principal component of Gram-negative bacteria that alerts the host to invading bacteria and triggers defensive responses (3, 4). These responses are usually beneficial and effective but may also become excessive and lead to endotoxic shock (3-5). Both LBP and BPI modulate the bioactivity of LPS (2, 3, 5). LBP is a plasma protein that catalyzes the transfer of LPS from LPS aggregates to other LPS-binding proteins (3, 6-9). Prominent among these is CD14, a surface molecule of myeloid cells that is also present in the circulation as a soluble protein. LBP and CD14 together represent the main pathway by which cells recognize low concentrations of LPS and are stimulated to respond to Gram-negative bacteria (3, 10, 11). In contrast to the LPS-stimulatory properties of LBP, binding of LPS by BPI results in inhibition of the bioactivities of LPS (2). BPI is produced by polymorphonuclear leukocytes and stored in its azurophilic granules (12, 13). It contributes substantially to both the intracellular and extracellular antibacterial activity of polymorphonuclear leukocyte-rich inflammatory exudates toward Gram-negative bacteria (14, 15). The high affinity of BPI
Journal of Biological Chemistry, 2003
Lipopolysaccharide (LPS)-binding protein (LBP) is an acute phase reactant that may play a dual ro... more Lipopolysaccharide (LPS)-binding protein (LBP) is an acute phase reactant that may play a dual role in vivo, both potentiating and decreasing cell responses to bacterial LPS. Whereas low concentrations of LBP potentiate cell stimulation by transferring LPS to CD14, high LBP concentrations inhibit cell responses to LPS. One inhibitory mechanism involves the ability of LBP to neutralize LPS by transferring it to plasma lipoproteins, whereas other inhibitory mechanisms, such as the one described here, do not require exogenous lipoproteins. Here we show that LBP can inhibit monocyte responses to LPS that has already bound to membrane-bound CD14 (mCD14) on the cell surface. LBP caused rapid dissociation of LPS from mCD14 as measured by the ability of LBP to inhibit cross-linking of a radioiodinated, photoactivatable LPS derivative to mCD14. Whereas LBP removed up to 75% of the mCD14-bound LPS in 10 min, this was not accompanied by extensive release of the LPS from the cells. The cross-linking data suggest that much of the LPS that remained bound to the cells was associated with LBP. The ability of LBP to inhibit cell responses could not be explained by its effect on LPS internalization, because LBP did not significantly increase the internalization of the cell-bound LPS. In cell-free LPS cross-linking experiments, LBP inhibited the transfer of LPS from soluble CD14 to soluble MD-2. Our data support the hypothesis that LBP can inhibit cell responses to LPS by inhibiting LPS transfer from mCD14 to the Toll-like receptor 4-MD-2 signaling receptor. Lipopolysaccharide (LPS 1 ; endotoxin), an abundant component of the outer membrane of Gram-negative bacteria, is one
Journal of Biological Chemistry, 1998
The hypothesis that soluble peptidoglycan (sPGN, a macrophage-activator from Gram-positive bacter... more 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 32 P-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 (K D ؍ 25 nM versus 41 nM). LPS-binding protein (LBP) increased the binding of sCD14 to sPGN by adding another lower affinity K D and another higher B max , but for ReLPS, LBP increased the affinity of binding by yielding two K D 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. Peptidoglycan (PGN) 1 is a polymer of alternating GlcNAc and MurNAc cross-linked by short peptides, present in the cell walls of all bacteria (1). PGN, similar to lipopolysaccharide (LPS) from the cell walls of Gram-negative bacteria, can reproduce most of the clinical manifestations of bacterial infections, including fever, acute-phase response,
Journal of Biological Chemistry, 1997
The stimulation of nonmyeloid cells by lipopolysaccharide (LPS) is mediated by the serum protein,... more The stimulation of nonmyeloid cells by lipopolysaccharide (LPS) is mediated by the serum protein, soluble CD14 (sCD14). We have examined the interaction of sCD14 with whole cells using a biologically active radiolabeled sCD14 molecule as a ligand. Specific binding of sCD14 to nonmyeloid cells is detected only when it is first incubated with both LPS and the serum LPS-binding protein (LBP). Through the use of an anti-CD14 monoclonal antibody, we demonstrate that sCD14 must interact with LPS in order for cellular binding to occur. Although LBP is traditionally known to function as a catalyst in the transfer of LPS to sCD14, our results reveal that LBP is actually a physical part of sCD14containing,cell-associatingcomplexes.TheLPS-andLBPdependent cell surface binding of sCD14 appears to be distinct from events leading to cell stimulation, since certain anti-CD14 and anti-LBP monoclonal antibodies have different effects on cellular binding versus cellular activation. Bound sCD14 is internalized, indicating that the LBP-and LPS-dependent binding of sCD14 may represent a novel general mechanism by which nonmyeloid cells clear LPS.
Journal of Biological Chemistry, 2001
Journal of Biological Chemistry, 2006
Toll-like receptor 4 and MD-2 form a receptor for lipopolysaccharide (LPS), a major constituent o... more Toll-like receptor 4 and MD-2 form a receptor for lipopolysaccharide (LPS), a major constituent of Gram-negative bacteria. MD-2 is a 20-25-kDa extracellular glycoprotein that binds to Tolllike receptor 4 (TLR4) and LPS and is a critical part of the LPS receptor. Here we have shown that the level of MD-2 expression regulates TLR4 activation by LPS. Using site-directed mutagenesis, we have found that glycosylation has no effect on MD-2 function as a membrane receptor for LPS. We used alanine-scanning mutagenesis to identify regions of human MD-2 that are important for TLR4 and LPS binding. We found that mutation in the N-terminal 46 amino acids of MD-2 did not substantially diminish LPS activation of Chinese hamster ovary (CHO) cells co-transfected with TLR4 and mutant MD-2. The residues 46-50 were important for LPS activation but not LPS binding. The residues 79-83, 121-124, and 125-129 are identified as important in LPS activation but not surface expression of membrane MD-2. The function of soluble MD-2 is somewhat more sensitive to mutation than membrane MD-2. Our results suggest that the 46-50 and 127-131 regions of soluble MD-2 bind to TLR4. The region 79-120 is not involved in LPS binding but affects monomerization of soluble MD-2 as well as TLR4 binding. We define the LPS binding region of monomeric soluble MD-2 as a cluster of basic residues 125-131. Studies on both membrane and soluble MD-2 suggest that domains of MD-2 for TLR4 and LPS binding are separate as well as overlapping. By mapping these regions on a three-dimensional model, we show the likely binding regions of MD-2 to TLR4 and LPS.
Immunity, 2006
CD14 is a well-known pattern-recognition receptor in the innate immune system. Here, we show that... more CD14 is a well-known pattern-recognition receptor in the innate immune system. Here, we show that CD14 enhances double-stranded RNA (dsRNA)-mediated Toll-like receptor 3 (TLR3) activation. Bone marrowderived macrophages (BMDMs) from CD14 2/2 mice exhibited impaired responses to polyinosine-polycytidylic acid (pIpC) and reduced production of inflammatory cytokines. CD14 2/2 mice injected with pIpC also showed impaired cytokine production. When tested with [ 32 P] labeled pIpC small fragments (pIpCsf) that maintain the inflammatory activity of crude pIpC, CD14 directly bound pIpCsf and mediated cellular uptake of pIpCsf. Our data show that TLR3 is intracellular and directly interacts with CD14. Internalized pIpCsf was localized in the lysosomes via the endosomes. In unstimulated cells, neither CD14 nor TLR3 was detected in the lysosomes. However, TLR3 was localized in the lysosomes as was CD14 once the cells took up pIpC. We also observed that internalized pIpCsf colocalized with CD14 and TLR3. Consequently, CD14 mediates pIpC uptake and enhances TLR3 signaling.
Clinical Infectious Diseases, 1999
Recent work has identified two proteins that work together to enable many cell types to respond t... more 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 LPSinitiated transmembrane signaling and internalization of LPS-protein complexes. This article is part of a series of papers presented at a symposium entitled from rabbit blood inhibited responses to LPS [21], that LBP ''Molecular Mechanisms of Microbial Host Cell Interactions in Periodontal enhanced the LPS-dependent activation of rabbit peritoneal Disease'' that was held on 14
Circulation Research, 2004
Innate immune system activation is associated with atherosclerotic lesion development. The specif... more Innate immune system activation is associated with atherosclerotic lesion development. The specific sites of lesion development are believed to be defined by the shear stress of blood flow. Consequently, we investigated the responsiveness of human coronary artery endothelial cells (HCAECs) to Toll-like receptor (TLR) 2 and 4 agonists in an in vitro model of chronic laminar flow. HCAECs under chronic laminar flow were found to be normally responsive to lipopolysaccharide (and tumor necrosis factor) in terms of E-selectin expression but were found to be hyporesponsive to stimulation with the specific TLR2 ligands macrophage activating lipopeptide-2, PAM 2 -Cys, and Lip19; this was observed to be attributable to downregulation of TLR2 transcription and protein expression. We found that laminar flow induced SP1 serine phosphorylation by protein kinase CK2 and thereby blocked SP1 binding to the TLR2 promoter, which is required for TLR2 expression. This regulatory mechanism also blocked l...
Biochemical Journal, 2005
SP-A (surfactant protein A) is a lipid-binding collectin primarily involved in innate lung immuni... more SP-A (surfactant protein A) is a lipid-binding collectin primarily involved in innate lung immunity. SP-A interacts with the bacterial rough LPS (lipopolysaccharide) Re-LPS (Re595 mutant of LPS from Salmonella minnesota), but not with smooth LPS. In the present study, we first examined the characteristics of the interaction of human SP-A with Re-LPS. Fluorescence intensity and anisotropy measurements of FITC-labelled Re-LPS in the presence and absence of SP-A indicated that SP-A bound to Re-LPS in solution in a Ca2+-independent manner, with a dissociation constant of 2.8×10−8 M. In the presence of calcium, a high-mobility complex of SP-A and [3H]Rb-LPS (Rb mutant of LPS from Escherichia coli strain LCD 25) micelles was formed, as detected by sucrose density gradients. Re-LPS aggregation induced by SP-A was further characterized by light scattering. On the other hand, human SP-A inhibited TNF-α (tumour necrosis factor-α) secretion by human macrophage-like U937 cells stimulated with e...