TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved in lipopolysaccharide-induced activation of the nuclear factor-B (NF-B) pathway, HEK 293 cells were transiently transfected with human TLR4 cDNA and an NF-B-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14 complexes. The results demonstrate that lipopolysaccharide stimulates NF-Bmediated gene expression in cells transfected with the TLR4 gene in a dose-and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocked TLR4-mediated transgene activation in a dosedependent manner (IC 50 ϳ30 nM). These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-B activation and subsequent cellular events. Lipopolysaccharide (LPS),1 a component of the outer membrane of Gram-negative bacteria, is a potent activator of a variety of mammalian cell types (1, 2). Activation by LPS constitutes the first step in a cascade of events believed to lead to the manifestation of Gram-negative sepsis, a condition that results in approximately 20,000 annual deaths in the United States (3). Activation of LPS-responsive cells, such as monocytes and macrophages, occurs rapidly after LPS interacts with circulating LPS-binding protein and CD14, a glycosylphosphatidylinositol-linked cell surface glycoprotein necessary for sensitive responses to LPS (1, 2). LPS has been shown to initiate multiple intracellular signaling events (4), including the activation of NF-B, which ultimately leads to the synthesis and release of a number of proinflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-␣ (1). However, since CD14 is not a transmembrane protein, it lacks the ability to transduce cytoplasmic signals (2), and before the recent discovery of Toll-like receptors (TLRs), the identity of a transmembrane protein that could relay LPS-induced signals across the cell-surface membrane remained elusive.Toll is a transmembrane receptor in Drosophila that is involved in dorsal-ventral patterning in embryos and in the induction of an anti-fungal response (5, 6). Activation of the Toll receptor by its ligand Spä tzle results in the interaction and stimulation of several signaling molecules that are homologous to proteins involved in NF-B activation by the IL-1 receptor in mammalian cells (7,8). The cloning of a family of human receptors structurally related to Drosophila Toll revealed five proteins that have extracellular domains that contain multiple leucine-rich repeats and cytoplasmic domains with sequence homology to the intracellular portion of the IL-1 receptor (9). Furthermore, constitutively active mutants of TLR2, TLR4, and TLR5 can induce the activation of NF-B (10, 11)...

Cellular fibronectin, which contains an alternatively spliced exon encoding type III repeat extra domain A (EDA), is produced in response to tissue injury. Fragments of fibronectin have been implicated in physiological and pathological processes, especially tissue remodeling associated with inflammation. Because EDAcontaining fibronectin fragments produce cellular responses similar to those provoked by bacterial lipopolysaccharide (LPS), we examined the ability of recombinant EDA to activate Toll-like receptor 4 (TLR4), the signaling receptor stimulated by LPS. We found that recombinant EDA, but not other recombinant fibronectin domains, activates human TLR4 expressed in a cell type (HEK 293 cells) that normally lacks this Toll-like receptor. EDA stimulation of TLR4 was dependent upon co-expression of MD-2, a TLR4 accessory protein. Unlike LPS, the activity of EDA was heat-sensitive and persisted in the presence of the LPS-binding antibiotic polymyxin B and a potent LPS antagonist, E5564, which completely suppressed LPS activation of TLR4. These observations provided a mechanism by which EDA-containing fibronectin fragments promote expression of genes involved in the inflammatory response.

Background Polychlorinated biphenyls (PCBs) are persistent environmental pollutants which are detectable in the serum of all American adults. Amongst PCB congeners, PCB 153 has the highest serum level. PCBs have been dose-dependently associated with obesity, metabolic syndrome, and nonalcoholic fatty liver disease (NAFLD) in epidemiological studies. Objective The purpose of this study is to determine mechanisms by which PCB 153 worsens diet-induced obesity and NAFLD in male mice fed a high fat diet (HFD). Methods Male C57BL6/J mice were fed either control or 42% milk fat diet for 12 weeks with or without PCB 153 co-exposure (50 mg/kg i.p. × 4). Glucose tolerance test was performed, and plasma and tissues were obtained at necropsy for measurements of adipocytokine levels, histology and gene expression. Results In control diet-fed mice, addition of PCB 153 had minimal effects on any of the measured parameters. However, PCB 153 treatment in high fat-fed mice was associated with increased visceral adiposity, hepatic steatosis and plasma adipokines including adiponectin, leptin, resistin and plasminogen activator inhibitor-1 levels. Likewise, co-exposure reduced expression of hepatic genes implicated in β-oxidation while increasing the expression of genes associated with lipid biosynthesis. Regardless of diet, PCB 153 had no effect on insulin resistance or tumor necrosis factor alpha levels. Conclusion PCB 153 is an obesogen which exacerbates hepatic steatosis; alters adipocytokines; and disrupts normal hepatic lipid metabolism when administered with HFD, but not control diet. Because all U.S. adults have been exposed to PCB 153, this particular nutrient-toxicant interaction potentially impacts human obesity/NAFLD.

Lipopolysaccharide (LPS) stimulates multiple signaling events, including nuclear factor-B (NF-B) activity and the mitogen-activated protein (MAP) kinases, ERK, JNK, and p38 in LPS-responsive cells, resulting in transcriptional activation and cytokine generation. LPS-induced signaling via toll-like receptor 4 (TLR4) results in the activation of the transcription factor NF-B. Since LPS activates other signaling cascades in responsive cells, the objective of this study was to determine whether such events are mediated by TLR4 in response to LPS. We generated human embryonic kidney cells (HEK293) that stably express TLR4 (HEK-TLR4) and examined their responsiveness to LPS by measuring NF-B activity and production of interleukin-8 (IL-8). A trans-reporting system was used to measure the activity of Elk-1, an ETS-domain transcription factor targeted by MAP kinase pathways. LPS stimulated NF-B reporter activity and IL-8 production but not Elk-1 activity in HEK-TLR4 cells. When MD-2, a protein associated with the extracellular domain of TLR4, was expressed in these cells, there was a marked increase in Elk-1 activity as well as ERK, JNK, and p38 MAP kinase phosphorylation in response to LPS. TLR4-mediated NF-B reporter activity and IL-8 production was enhanced by the expression of MD-2. This study demonstrates that expression of both TLR4 and MD-2 is required for LPS to activate or augment the MAP kinase pathways, Elk-1 stimulation, and IL-8 generation.The pathophysiology of Gram-negative sepsis and septic shock is caused by lipopolysaccharide (LPS), 1 a component of the outer membrane of bacteria that can activate a variety of mammalian cell types, including monocytes and macrophages (1, 2). This, in turn, causes the release of a number of proinflammatory mediators, such as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor-␣ (1). Activation of LPS-responsive cells occurs rapidly after LPS interacts with circulating LPSbinding protein and CD14, a glycosylphosphatidylinositollinked cell surface or soluble protein necessary for efficient responses to LPS (2). Evidence from studies in vitro indicates that expression of TLR2 and TLR4, two members of the toll-like receptor (TLR) family of cell surface proteins, can enable the activation of nuclear factor-B (NF-B) and expression of genes for IL-1, 4). TLR4 has been shown to mediate LPS-induced activation of NF-B in human embryonic kidney cells (HEK293) (5, 6), whereas in 293T and Ba/F3 cells, TLR4-mediated responses to LPS requires the presence of MD-2, a cell surface protein that associates with TLR4 (7). Observations from studies with TLR2 and TLR4 knockout mice indicate that TLR4, but not TLR2, is necessary for responses to LPS under physiological conditions; TLR2 knockout mice respond normally to LPS, whereas TLR4 knockout mice or spontaneous TLR4 mutants (C3H/HeJ and C57/10ScCr) are not responsive to LPS (8 -10). On the other hand, studies with TLR2 knockout mice suggest that TLR2 has a broader recognition pattern than TLR4, which extends to a number of different...

Recent epidemiological studies suggest that traffic-related air pollution may have detrimental effects on cardiovascular health. Previous studies reveal that gasoline emissions can induce several enzyme pathways involved in the formation and development of atherosclerotic plaques. As a direct comparison, the present study examined the impact of diesel engine emissions on these pathways, and further examined the effects on vascular lesion pathology. Apolipoprotein E-null mice were simultaneously placed on a high fat chow diet and exposed to four concentrations, plus a high concentration exposure with particulates (PM) removed by filtration, of diesel emissions for 6 h/d for 50 days. Aortas were subsequently assayed for alteration in matrix metalloproteinase-9, endothelin-1, and several other biomarkers. Diesel induced dose-related alterations in gene markers of vascular remodeling and aortic lipid peroxidation; filtration of PM did not significantly alter these vascular responses, indicating that the gaseous portion of the exhaust was a principal driver. Immunohistochemical analysis of aortic leaflet sections revealed no net increase in lesion area, but a significant decrease in lipid-rich regions and increasing trends in macrophage accumulation and collagen content, suggesting that plaques were advanced to a more fragile, potentially more vulnerable state by diesel exhaust exposure. Combined with previous studies, these results indicate that whole emissions from mobile sources may have a significant role in promoting chronic vascular disease.