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PubMed2006

Differential effects of Gram-positive versus Gram-negative bacteria on NOSII and TNFalpha in macrophages: role of TLRs in synergy between the two.

1. Gram-negative and Gram-positive bacteria are sensed by Toll-like receptor (TLR)4 and TLR2, respectively. TLR4 recruits MyD88 and TRIF, whereas TLR2 recruits MyD88 without TRIF. NOSII and TNFalpha are central genes in innate immunity and are thought to be differentially regulated by the MyD88 versus TRIF signalling pathways. Here, we have used Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli and highly selective TLR ligands to establish the precise relationship between TLR2, TLR1, TLR6 and TLR4 for NOSII versus TNFalpha induction. 2. In murine macrophages at 24 h, E. coli or LPS (TLR4) induced NO and TNFalpha release. In contrast, S. aureus (TLR2/TLR1/TLR6) or Pam(3)CSK4 (TLR2/TLR1), or FSL-1 and LTA (TLR2/TLR6) induced TNFalpha without an effect on NO. 3. At later time points (48-72 h), S. aureus induced NO release. The ability of S. aureus, but not E. coli or LPS, to induce NO release was inhibited by anti-TNFalpha-binding antibodies. 4. At 24 h, LPS synergised with TLR2 ligands to induce NO release and NOSII protein expression. LPS also induced the expression of TLR2 gene expression without affecting levels of TLR4. 5. Using cells from TLR2(-/-) or TLR4(-/-) mice, the ability of LPS to synergise with S. aureus or Pam(3)CSK4 was found to be dependent on both TLR2 and TLR4. 6. These observations are the first to clearly delineate the role of separately activating TLR2 and TLR4 in the induction of NOSII and TNFalpha genes compared with their coinduction when both receptor pathways are activated.

Animals↗
PubMed2007

Tyrosine kinase Syk associates with toll-like receptor 4 and regulates signaling in human monocytic cells.

Toll-like receptor 4 (TLR4) induces an innate immune response in mammals by recognizing lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria. In this study, we show that tyrosine kinase Syk constitutively associates with TLR4 in THP-1 cells. As previously reported in peripheral blood mononuclear cells, TLR4 gets inducibly tyrosine phosphorylated upon LPS engagement in THP-1 cells. Piceatannol, a pharmacological inhibitor of the tyrosine kinase Syk, abrogates TLR4 tyrosine phosphorylation at low doses. The kinetics of TLR4 tyrosine phosphorylation in THP-1 cells coincides with an early wave of Syk tyrosine phosphorylation. Additionally, serine threonine kinase interleukin-1 (IL1) receptor-associated kinase 1 (IRAK-1) is transiently recruited to the complex containing adaptor molecule MyD88, TLR4 and Syk within 1 min of LPS engagement and dissociates by 30 min. Finally, the inhibition of Syk with piceatannol has no effect on LPS-mediated release of cytokines IL6, IL1beta, tumor necrosis factor-alpha, neither on chemokines macrophage inhibitory protein (MIP)1alpha, MIP1beta, monocyte chemoattractant protein -1, IL8, Groalpha and RANTES. However, IL10 and IL12p40 releases are significantly inhibited. Our findings implicate Syk as a novel modulator of LPS-mediated TLR4 responses in human monocytic cells and shed insight into the kinetics of early complex formation upon LPS engagement.

Cell Line, Tumor↗
PubMed2006

Reduced expression of Toll-like receptor 4 contributes to impaired cytokine response of monocytes in uremic patients.

Toll-like receptors (TLRs) play a pivotal role in pathogen recognition and subsequent cytokine synthesis by immune cells. Uremic patients have a high infectious morbidity, but it remains unclear if this arises from the defective innate immune responses related to TLRs. We studied TLR4 expression in monocytes and their intracellular cytokine synthesis in response to lipopolysaccharide (LPS) stimulation in 35 predialysis patients with chronic kidney disease (CKD) with or without predisposition to bacterial infections and 16 age-matched controls. Expression of TLR4 in unstimulated peripheral monocytes was determined by staining with anti-TLR4 antibody and analysis with flow cytometry. Monocytes were then stimulated by LPS, labeled with anti-CD14 antibody, and subjected to intracellular cytokine staining and flow cytometry. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and IL-8 synthesis was examined in CD14(+) monocytes. TLR4 expression was constitutively diminished in CKD patients with reduced expression being more severe in those CKD patients who were predisposed to infections. Monocytes from these infection prone CKD patients exhibited significantly reduced synthesis of TNF-alpha, IL-1beta, IL-6, and IL-8 in response to LPS challenge compared with those from control subjects. The intensity of synthesis of each cytokine significantly correlated with TLR4 expression levels in monocytes (P<0.01). The capacity of monocytes to synthesize proinflammatory cytokines was significantly reduced in infection prone CKD patients, and this may possibly be due to the reduced monocyte expression of TLR4. Abnormal TLR4 expression by monocytes may play a role in the susceptibility of such patients to bacterial infections.

Aged↗
PubMed2001

Toll-like receptor 4 is involved in the mechanism of early alcohol-induced liver injury in mice.

Chronic alcohol administration increases gut-derived endotoxin in the portal blood, which activates Kupffer cells and causes liver injury. Mice (C3H/HeJ) with mutations in toll-like receptor 4 (TLR4) are hyporesponsive to endotoxin. To test the hypothesis that TLR4 is involved in early alcohol-induced liver injury, the long-term intragastric ethanol feeding protocol developed by Tsukamoto and French for rats was adapted to mice. Animals with nonfunctional TLR4 and wild-type mice (C3H/HeOuJ) were compared. Two-month-old female mice were fed a high-fat liquid diet with either ethanol or isocaloric maltose-dextrin as control continuously for 4 weeks. There was no difference in mean urine alcohol concentrations between the groups. Dietary alcohol significantly increased liver-to-body weight ratios and serum alanine transaminase (ALT) levels in wild-type mice (109 +/- 18 U/L) over high-fat controls (40 +/- 3 U/L), effects that were blunted significantly in mice with a mutation of TLR4 (55 +/- 9 U/L). While no significant pathologic changes were observed in high-fat controls, dietary ethanol caused steatosis, mild inflammation, and focal necrosis in wild-type animals (pathology score = 5.2 +/- 1.2). These pathologic changes were significantly lower in TLR4-deficient mice fed ethanol (score = 2.0 +/- 1.3). Endotoxin levels in the portal vein were increased significantly after 4 weeks in both groups fed ethanol. Moreover, ethanol increased tumor necrosis factor alpha (TNF-alpha) mRNA expression in wild-type, but not in TLR4-deficient, mice. These data are consistent with the hypothesis that Kupffer cell activation by endotoxin via TLR4 is involved in early alcohol-induced liver injury.

Alanine Transaminase↗
PubMed2005

The CNS role of Toll-like receptor 4 in innate neuroimmunity and painful neuropathy.

Neuropathic pain remains a prevalent and persistent clinical problem because of our incomplete understanding of its pathogenesis. This study demonstrates for the first time, to our knowledge, a critical role for CNS innate immunity by means of microglial Toll-like receptor 4 (TLR4) in the induction phase of behavioral hypersensitivity in a mouse and rat model of neuropathy. We hypothesized that after L5 nerve transection, CNS neuroimmune activation and subsequent cytokine expression are triggered by the stimulation of microglial membrane-bound TLR4. To test this hypothesis, experiments were undertaken to assess tactile and thermal hypersensitivity in genetically altered (i.e., TLR4 knockout and point-mutant) mice after L5 nerve transection. In a complementary study, TLR4 antisense oligodeoxynucleotide (ODN) was administered intrathecally to L5 spinal nerve injured rats to reduce the expression of spinal TLR4. Both the genetically altered mice and the rats treated with TLR4 antisense ODN displayed significantly attenuated behavioral hypersensitivity and decreased expression of spinal microglial markers and proinflammatory cytokines as compared with their respective control groups. This finding shows that TLR4 contributes to the initiation of CNS neuroimmune activation after L5 nerve transection. Further understanding of this early, specific, innate CNS/microglial response and how it leads to sustained glial/neuronal hypersensitivity may point to new therapies for the prevention and treatment of neuropathic pain syndromes.

Animals↗
PubMed2006

Combinatorial pattern recognition receptor signaling alters the balance of life and death in macrophages.

Macrophage pattern recognition receptors (PRRs) play key roles in innate immunity, but they also may contribute to disease processes under certain pathological conditions. We recently showed that engagement of the type A scavenger receptor (SRA), a PRR, triggers JNK-dependent apoptosis in endoplasmic reticulum (ER)-stressed macrophages. In advanced atherosclerotic lesions, the SRA, activated JNK, and ER stress are observed in macrophages, and macrophage death in advanced atheromata leads to plaque necrosis. Herein, we show that SRA ligands trigger apoptosis in ER-stressed macrophages by cooperating with another PRR, Toll-like receptor 4 (TLR4), to redirect TLR4 signaling from prosurvival to proapoptotic. Common SRA ligands activate both TLR4 signaling and engage the SRA. The TLR4 effect results in activation of the proapoptotic MyD88-JNK branch of TLR4, whereas the SRA effect silences the prosurvival IRF-3-IFN-beta branch of TLR4. The normal cell-survival effect of LPS-induced TLR4 activation is converted into an apoptosis response by immunoneutralization of IFN-beta, and the apoptosis effect of SRA ligands is converted into a cell-survival response by reconstitution with IFN-beta. Thus, combinatorial signaling between two distinct PRRs results in a functional outcome-macrophage apoptosis that does not occur with either PRR alone. PRR-induced macrophage death may play important roles in advanced atherosclerosis and in other innate immunity-related processes in which the balance between macrophage survival and death is critical.

Animals↗
PubMed2003

Toll-like receptor 4 on stromal and hematopoietic cells mediates innate resistance to uropathogenic Escherichia coli.

Innate host defenses at mucosal surfaces are critical in the early stages of many bacterial infections. In addition to cells of the traditional innate immune system, epithelial cells can also produce inflammatory mediators during an infection. However, the role of the epithelium in innate host defense in vivo is unclear. Recent studies have shown that lipopolysaccharide (LPS) recognition is critical for bladder epithelial cells to recognize and respond to Escherichia coli. Moreover, the LPS-nonresponsive mouse strain C3HHeJ, which has a mutation in the primary LPS receptor, Toll-like receptor 4 (TLR4), is extremely susceptible to infection with uropathogenic strains of E. coli. In this study, a bone marrow transplant approach was used to investigate the specific contributions of the bladder epithelium (and other stromal cells) in the TLR4-mediated innate immune response to the invading E. coli pathogen. Mice expressing the mutant TLR4 in the epithelialstromal compartment were not able to mount a protective inflammatory response to control the early infection even when their hematopoietic cells expressed wild-type TLR4. However, the presence of TLR4(+) epithelialstromal cells was not sufficient to activate an acute inflammatory response unless the hematopoietic cells were also TLR4(+). These results demonstrated that bladder epithelial cells play a critical role in TLR4-mediated innate immunity in vivo during a mucosal bacterial infection.

Animals↗
PubMed2000

PU.1 and interferon consensus sequence-binding protein regulate the myeloid expression of the human Toll-like receptor 4 gene.

The protein product of the Toll-like receptor (TLR) 4 gene has been implicated in the signal transduction events induced by lipopolysaccharide (LPS). In mice, destructive mutations of Tlr4 impede the normal response to LPS and cause a high susceptibility to Gram-negative infection. Expression of TLR4 mRNA in humans is restricted to a small number of cell types, including LPS-responsive myeloid cells, B-cells, and endothelial cells. To investigate the molecular basis for TLR4 expression in cells of myeloid origin, we cloned the human TLR4 gene and analyzed its putative 5'-proximal promoter. In transient transfections a region of only 75 base pairs upstream of the major transcription initiation site was sufficient to induce maximal luciferase activity in THP-1 cells. The sequence of this region is similar in human and mouse TLR4 genes and lacks a TATA box, typical Sp1-sites or CCAAT box sequences. Instead, it contains consensus-binding sites for Ets family transcription factors, octamer-binding factors, and a composite interferon response factor/Ets motif. The activity of the promoter in macrophages was strictly dependent on the integrity of both half sites of the composite interferon response factor/Ets motif, which was constitutively bound by the myeloid and B-cell-specific transcription factor PU.1 and interferon consensus sequence-binding protein. These results indicate that the two tissue-restricted transcription factors PU.1 and interferon consensus sequence-binding protein participate in the basal regulation of human TLR4 in myeloid cells. Cloning of the human TLR4 gene provides a basis for further investigation of the possible impact of genetic variations on the susceptibility to infection and sepsis.

Amino Acid Sequence↗
PubMed2003

Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to Toll-like receptor-4 aggregation and signal transduction.

Three cell-surface proteins have been recognized as components of the mammalian signaling receptor for bacterial lipopolysaccharide (LPS): CD14, Toll-like receptor-4 (TLR4), and MD-2. Biochemical and visual studies shown here demonstrate that the role of CD14 in signal transduction is to enhance LPS binding to MD-2, although its expression is not essential for cellular activation. These studies clarify how MD-2 functions: we found that MD-2 enables TLR4 binding to LPS and allows the formation of stable receptor complexes. MD-2 must be bound to TLR4 on the cell surface before binding can occur. Consequently, TLR4 clusters into receptosomes (many of which are massive) that recruit intracellular toll/IL-1/resistance domain-containing adapter proteins within minutes, thus initiating signal transduction. TLR4 activation correlates with the ability of MD-2 to bind LPS, as MD-2 mutants that still bind TLR4, but are impaired in the ability to bind LPS, conferred a greatly blunted LPS response. These findings help clarify the earliest events of TLR4 triggering by LPS and identify MD-2 as an attractive target for pharmacological intervention in endotoxin-mediated diseases.

Amino Acid Sequence↗
PubMed2003

Cell distributions and functions of Toll-like receptor 4 studied by fluorescent gene constructs.

Bacterial lipopolysaccharide (LPS) is recognized in mammals by a receptor complex composed of CD14, Toll-like receptor 4 (TLR4), and MD-2. The detailed mechanisms of how TLR4 transmits the signal from the outside to the inside of the cell remain to be elucidated. One way of studying TLR4 signaling mechanisms is to construct chimeras of TLR molecules C-terminally fused to fluorescent proteins and stably express these constructs in cells. Such constructs are functional when transfected into HEK293 epithelial cells. Confocal microscopy of TLR4 expression in live cells demonstrated pronounced expression on the plasma membrane as well in the Golgi apparatus. Studies were performed to clarify whether expression of TLR4 in the Golgi was necessary for LPS stimulation. Rapid recycling of TLR4/CD14/MD-2 complexes between the Golgi and the plasma membrane was a prominent phenomenon. In agreement with other types of plasma membrane receptors, aggregation of TLR4 by immobilized TLR4 antibodies was sufficient to induce signaling. Also, pharmacological disruption of the Golgi did not inhibit LPS induced NF-kappaB activation. Furthermore, LPS stimulation recruited the adapter molecule, MyD88, to the inside of the plasma membrane. Thus, LPS signaling commences on the plasma membrane and is independent of trafficking to the Golgi.

Animals↗
PubMed1999

MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4.

Toll-like receptor 4 (TLR4) is a mammalian homologue of Drosophila Toll, a leucine-rich repeat molecule that can trigger innate responses against pathogens. The TLR4 gene has recently been shown to be mutated in C3H/HeJ and C57BL/10ScCr mice, both of which are low responders to lipopolysaccharide (LPS). TLR4 may be a long-sought receptor for LPS. However, transfection of TLR4 does not confer LPS responsiveness on a recipient cell line, suggesting a requirement for an additional molecule. Here, we report that a novel molecule, MD-2, is requisite for LPS signaling of TLR4. MD-2 is physically associated with TLR4 on the cell surface and confers responsiveness to LPS. MD-2 is thus a link between TLR4 and LPS signaling. Identification of this new receptor complex has potential implications for understanding host defense, as well as pathophysiologic, mechanisms.

Animals↗
PubMed2002

Toll-like receptor 4 is not involved in host defense against pulmonary Legionella pneumophila infection in a mouse model.

Legionella pneumophila is a gram-negative microorganism that causes a severe pneumonia known as "legionnaires disease." Toll-like receptor 4 (TLR4) transduces the lipopolysaccharide signal and is therefore considered to play a role in host defense against gram-negative bacterial infection. To determine the role of TLR4 in L. pneumophila pneumonia, C3H/HeJ mice, which display a nonfunctional gene encoding TLR4 (TLR4), and wild-type (wt) C3H/HeN mice were intranasally inoculated with L. pneumophila serogroup 1. Infection proceeded in an identical way in TLR4 mutant and wt mice, as reflected by similar bacterial outgrowth in the lungs. In addition, the inflammatory responses to L. pneumophila infection-as assessed by histopathologic analysis, cell influx in bronchoalveolar lavage fluid, myeloperoxidase activity in lungs, and lung cytokine concentrations-were indistinguishable in TLR4 mutant and wt mice. These data suggest that, in this mouse model, TLR4 does not play a role in resistance to L. pneumophila.

Animals↗
PubMed2004

Toll-like receptor 4 plays a protective role in pulmonary tuberculosis in mice.

Toll-like receptors (TLR) play an essential role in the innate recognition of microorganisms by the host. To determine the role of TLR4 in host defense against lung tuberculosis, TLR4 mutant (C3H/HeJ) and wild-type (C3H/HeN) mice were intranasally infected with live Mycobacterium tuberculosis. TLR4 mutant mice were more susceptible to pulmonary tuberculosis, as indicated by a reduced survival and an enhanced mycobacterial outgrowth. Lung infiltrates were more profound in TLR4 mutant mice and contained more activated T cells. Splenocytes of infected TLR4 mutant mice demonstrated a reduced capacity to produce the protective type 1 cytokine IFN-gamma upon antigen-specific stimulation, indicating that TLR4 may be involved in the generation of acquired T cell-mediated immunity. These data suggest that TLR4 plays a protective role in host defense against lung infection by M. tuberculosis.

Animals↗
PubMed2005

Conditional signaling by Toll-like receptor 4.

Signaling through Toll-like receptor 4 (TLR4) is thought to initiate innate and adaptive immune responses. Signaling of TLR4 is usually studied using isolated cells, which are activated by sub-nanomolar concentrations of lipopolysaccharide (LPS). However, in normal tissues, cells bearing TLR4 reside in microenvironments containing large amounts of endogenous substances that can stimulate the receptor. We developed an in vitro model system using the human cell line HEK 293 and an in vivo model using mice that have normal or that lack TLR4 receptors to study how TLR4 functions in such microenvironments. Here we report that signaling through TLR4 is strongly inhibited by intact extracellular matrix and that inhibition is abrogated and endogenous agonist(s) are liberated when the matrix is degraded. Thus, release from inhibition rather than direct stimulation by agonists such as LPS is the critical first event by which TLR4 initiates immune responses.

Animals↗
PubMed2004

Role of Toll-like receptor 4 on pancreatic and pulmonary injury in a mice model of acute pancreatitis associated with endotoxemia.

OBJECTIVE: Infection of pancreatic necrosis is a severe complication of acute pancreatitis. Because Toll-like receptor 4 (TLR4) has been identified as a receptor necessary to transduct the signal of bacteria-derived lipopolysaccharide into cells, we investigated the role of TLR4 on pancreatic and pulmonary injury in acute pancreatitis and acute pancreatitis associated with endotoxemia in wild-type and TLR4-deficient mice. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: Heterozygous TLR4 mice. INTERVENTIONS: Mice were injected intraperitoneally with a supramaximal dose of cerulein each hour for 10 hrs. To mimic infection, additional groups of mice were injected with lipopolysaccharide in the presence or absence of cerulein injections. MEASUREMENTS AND MAIN RESULTS: The severity of acute pancreatitis was assessed by serum amylase activity, pancreatic edema, acinar cell necrosis, and pancreas myeloperoxidase activity. Lung injury was quantitated by lung microvascular permeability and lung myeloperoxidase activity. Injections of cerulein induced an edematous pancreatitis that was of similar severity in wild-type and TLR4-deficient mice. Lipopolysaccharide alone had no toxic effect on pancreas and lungs and did not worsen the pancreatic injury induced by cerulein in wild-type and TRL4-deficient mice. In contrast, lipopolysaccharide worsened pancreatitis-associated lung injury, and the deficiency in TLR4 fully prevented this aggravation. CONCLUSIONS: TLR4 may not play a role in the pancreatitis-associated lung injury but participates in the pulmonary injury mediated by endotoxemia.

Acute Disease↗
PubMed2005

Toll-like receptor 4 deficiency and acute pancreatitis act similarly in reducing host defense during murine Escherichia coli peritonitis.

OBJECTIVE: Acute pancreatitis is frequently complicated by Gram-negative sepsis. Mammalian cells recognize lipopolysaccharide from Gram-negative bacteria via Toll-like receptor (TLR) 4. The objective of this study was to determine the role of TLR4 in the defense against Gram-negative sepsis in previously healthy mice and in animals with preexisting pancreatitis. DESIGN: A controlled, in vivo laboratory study. SETTING: Research laboratory of a health sciences university. SUBJECTS: Female C3H/HeJ (nonfunctional TLR4 mutant) and C3H/HeN (wild-type) mice. INTERVENTIONS: Abdominal sepsis was induced by the intraperitoneal injection of Escherichia coli. Pancreatitis was induced by 12 hourly intraperitoneal injections of cerulein. MEASUREMENTS AND MAIN RESULTS: The following experiments were performed. First, healthy TLR4 mutant mice demonstrated an enhanced bacterial load and dissemination of the infection relative to wild-type mice after intraperitoneal injection with E. coli, associated with a reduced early release of proinflammatory cytokines and an attenuated influx of neutrophils into the peritoneal fluid. Second, wild-type mice in which acute pancreatitis was induced by repeated cerulein injections showed an increased bacterial load and dissemination of E. coli relative to wild-type mice without pancreatitis, which was accompanied by a blunted proinflammatory cytokine response by peritoneal macrophages ex vivo and a diminished early cytokine and neutrophil response in vivo. Third, whereas the severity of cerulein-induced pancreatitis was similar in TLR4 mutant and wild-type mice, the important contribution of TLR4 to an effective host defense against E. coli sepsis observed in previously healthy mice was no longer present in mice with preexisting pancreatitis. CONCLUSIONS: These data suggest that TLR4 deficiency and acute pancreatitis act similarly in reducing host defense against E. coli peritonitis and that the role of TLR4 in severe Gram-negative infection depends, at least in part, on the presence of preexisting critical illness.

Acute Disease↗
PubMed2005

Roles of endotoxin-related signaling molecules in the progression of acute necrotizing pancreatitis in mice.

OBJECTIVE: To study the potential roles of lipopolysaccharide (LPS) signaling molecules, LPS-binding protein (LBP), CD14, and Toll-like receptor 4 (TLR4) in mice with acute necrotizing pancreatitis (ANP). METHODS: The ANP model was made by 7 intraperitoneal injections of cerulein (50 mug/kg) at hourly intervals, challenged by LPS administration of a dose of 5 mg/kg intraperitoneally 5 hours after the first injection of cerulein. Fifty-nine Balb/C mice were divided into 4 groups: group A, ANP control group, n = 18, received physiological saline; group B, anti-LBP group, n = 18, received 200 mug anti-LBP antibody; group C, anti-CD14 group, n = 18, received 20 microg anti-CD14 antibody; group D, anti-TLR4 group, n = 5, received 20 mug anti-TLR4 antibody. All treatments were given at 15 minutes before LPS injection. Serum amylase and lactic dehydrogenase (LDH) were measured. Histologic alteration of the pancreas was evaluated. Expressions of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and intercellular adhesion molecular (ICAM-1) mRNA were determined using reverse transcription polymerase chain reaction (RT-PCR). Myeloperoxidase (MPO) activity in pancreas was also analyzed. Nuclear factor-kappaB (NF-kappaB) p65 subunit was measured by immunohistochemistry and Western blotting. RESULTS: Pretreatment of animals with anti-CD14 antibody resulted in a significant decrease in serum amylase and LDH levels, reduction of the severity of pancreatic injury, down-regulation of TNF-alpha, IL-1beta, and ICAM-1 mRNA expression, decrease in pancreatic MPO activity, and decrease in NF-kappaB expression compared with ANP control mice. In contrast, mice pretreated with anti-LBP antibody or anti-TLR4 antibody resulted in increasing of serum amylase and LDH levels, aggravation of the severity of necrosis and inflammation in pancreas, up-regulation of TNF-alpha, IL-1beta, and ICAM-1 mRNA expression, increasing of pancreatic MPO activity, and up-regulation of NF-kappaB expression compared with ANP control mice. CONCLUSIONS: (1) LBP per se possesses a protective effect on ANP. It could facilitate clearance of endotoxin. (2) CD14 plays a crucial intermediate role in the progression of ANP. (3) TLR4, the essential transducer of LPS responses, may act independently of LPS. The antibody against TLR4 may mimic endotoxin and induce activation of downstream cytokines. For implication, recombinant LBP, anti-CD14 antibody, or silent TLR4 might alleviate the progression of ANP.

Acute-Phase Proteins↗
PubMed2006

Pre- and postoperative cytokine release after in vitro whole blood lipopolysaccharide stimulation and frequent toll-like receptor 4 polymorphisms.

In a prospective cohort study, we examined 62 patients undergoing major surgical cancer therapy for Toll-like receptor 4 (TLR4) gene polymorphisms (Asp299Gly and Thr399Ile) and their influence on cytokine levels pre- and postoperatively, as well as cytokine levels after whole blood lipopolysaccharide (LPS) stimulation. Incidence of the TLR4 gene single nucleotide polymorphism (SNP) Asp299Gly/Thr399Ile was 14.5% (9/62). Overall, mortality was unaffected by the TLR4 SNP. Preoperative cytokine levels were low, with most of the values of cytokines being below the detection levels. After preoperative stimulation of whole blood with 50 pg/mL LPS, TNF-alpha and IL-6 values increased significantly in both groups. However, no significant influence was detectable between the TLR4 SNP group and the wild type group (WT group). Postoperative IL-6 levels, but not TNF-alpha levels, were significantly increased in both groups. Postoperative LPS stimulation resulted in significantly lower TNF-alpha levels compared with preoperative induction, with a more than 2.3-fold decrease in the TLR4 SNP group: 310.83 pg/mL (SD: 117.53) to 134.08 pg/mL (SD: 91.49; P < 0.001) and a 2.2-fold decrease in the WT group: 422.97 pg/mL (SD: 662.57) to 191.68 pg/mL (SD:147.26; P = 0.031). IL-6 levels after stimulation were comparably decreased with similarly no significant difference between the two groups. We conclude that the TLR4 polymorphism Asp299Gly/Thr399Ile has no influence on cytokine release after LPS stimulation in the early and late course after major surgery. The LPS adaptation effect of cytokine release after surgery is furthermore not affected by the presence of the TLR4 polymorphism Asp299Gly/Thr399Ile.

Amino Acid Substitution↗