Lipopolysaccharide Receptor Expression Research Articles

Influences of aging on expression of lipopolysaccharide receptor of kupffer cells in mice

Rationale: The hepatic immunity generally declines with aging. Clinically, the consequences of impaired hepatic immune function in the elderly include an increased susceptibility to infections. In the previous study, phagocytic function of Kupffer cells (resident hepatic macrophages) declines with aging. However, the influence of aging on expression of lipopolysaccharide (LPS) receptor’s expression of Kupffer cells is not clarified. Herein, we examined how the aging affects expression of LPS receptor (CD14) of Kupffer cells with mice model.

Involvement of lipopolysaccharide in ovarian cystic follicles in dairy cow: Expressions of LPS receptors and steroidogenesis-related genes in follicular cells of cystic follicles

In ovarian cystic follicles, molecular changes in the growing follicle may have a local action and contribute to anovulation and cystic formation. One of the candidate molecules that affect the steroid and gonadotropin signaling systems of cystic follicles is lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria. To examine the molecular characteristics of bovine cystic follicles, we analyzed LPS concentration in follicular fluid of cystic follicles, and the expression of LPS receptors and steroidogenesis-related genes in granulosa and theca cells. Cystic follicles were categorized as estradiol (E2)-active (EACF) and E2-inactive cystic follicle (EICF). Overall, LPS concentration in follicular fluid of EACF and EICF was higher compared with healthy preovulatory follicles (POF). Expression of luteinizing hormone receptor (LHR) in granulosa and thecal cells was decreased in EACF and EICF compared with POF. Expression of CYP19 in granulosa cells of EACF and EICF was lower than POF. High expression of StAR in granulosa and thecal cells was observed in EICF. In granulosa cells, the expression of TLR4 and TLR2 mRNA was higher in EICF than other follicles. By contrast, higher expression of TLR2 in thecal cells was observed in EICF. Thus, high LPS concentration in follicular fluid of cystic follicles may be associated with the regulation of expression of steroidogenesis-related genes in granulosa and theca cells. These finding revealed the molecular characteristics of bovine ovarian cysts and possible involvement of LPS in the pathology of cystic follicle diseases.

Lipid raft-dependent endocytosis negatively regulates responsiveness of J774 macrophage-like cells to LPS by down regulating the cell surface expression of LPS receptors

Acting through CD14 and TLR4/MD-2, lipopolysaccharide (LPS) triggers strong pro-inflammatory activation of macrophages, which, if not appropriately controlled, may lead to lethal septic shock. Therefore, numerous mechanisms of negative regulation of responses to LPS exist, but whether they include down-regulation of LPS receptors is not clear. We have found that in J774 cells, the clathrin-dependent endocytic pathway enables activation of TRIF-dependent TLR4 signaling within endosomes, but is not associated with the down-regulation of TLR4 or CD14 surface expression. In contrast, lipid raft-dependent endocytosis negatively regulates the basal cell surface expression of LPS receptors and, consequently, responsiveness to LPS. Together with observations that treatments, known to selectively disrupt lipid rafts, do not inhibit LPS-stimulated cytokine production, our results suggest that lipid rafts may serve as sites in which LPS receptors are sorted for endocytosis, rather than being platforms for the assembly of TLR4-centered signaling complexes, as suggested previously.

Granulocyte Colony Stimulating Factor Induces Lipopolysaccharide (LPS) Sensitization via Upregulation of LPS Binding Protein in Rat

Liver is the main organ for lipopolysaccharide (LPS) clearance. Sensitization to LPS is associated with the upregulation of LPS-binding protein (LBP) in animal models. Therefore, we hypothesized that LBP could induce LPS sensitization through enhancing hepatic uptake of LPS. In this study, we examined the role of LBP in pathogenesis of LPS induced systemic inflammatory response syndrome (SIRS). LBP expression was upregulated after granulocyte colony stimulating (G-CSF) pretreatment. The effect of LBP was further confirmed by blockade of LBP using LBP blocking peptide – LBPK95A. After G-CSF pretreatment, upregulation of LBP was observed in bone marrow cells and liver. The G-CSF induced LBP upregulation caused LPS hypersensitization in rats as indicated by higher mortality and severer liver damage. Of note, LBP blockade increased the survival rate and attenuated the liver injury. The LBP induced LPS hypersensitization was associated with increased hepatic uptake of LPS and augmented hepatic expression of LPS receptors, such as toll-like receptor (TLR)-4. Furthermore, LBP mediated early neutrophil infiltration, which led to increased monocyte recruitment in liver after LPS administration. In conclusion, G-CSF induced LBP expression could serve as a new model for investigation of LPS sensitization. We demonstrated the crucial role of LBP upregulation in pathogenesis of LPS induced SIRS.

Modulation of lipopolysaccharide receptor expression by lactate dehydrogenase-elevating virus

Lactate dehydrogenase-elevating virus (LDV) exacerbates mouse susceptibility to endotoxin shock through enhanced tumour necrosis factor (TNF) production by macrophages exposed to lipopolysaccharide (LPS). However, the in vivo enhancement of TNF production in response to LPS induced by the virus largely exceeds that found in vitro with cells derived from infected animals. Infection was followed by a moderate increase of Toll-like receptor (TLR)-4/MD2, but not of membrane CD14 expression on peritoneal macrophages. Peritoneal macrophages from LDV-infected mice unresponsive to type I interferons (IFNs) did not show enhanced expression of TLR-4/MD2 nor of CD14, and did not produce more TNF in response to LPS than cells from infected normal counterparts, although the in vivo response of these animals to LPS was strongly enhanced. In contrast, the virus triggered a sharp increase of soluble CD14 and of LPS-binding protein serum levels in normal mice. However, production of these LPS soluble receptors was similar in LDV-infected type I IFN-receptor deficient mice and in their normal counterparts. Moreover, serum of LDV-infected mice that contained these soluble receptors had little effect if any on cell response to LPS. These results suggest that enhanced response of LDV-infected mice to LPS results mostly from mechanisms independent of LPS receptor expression.

Altered biliary epithelial cell and monocyte responses to lipopolysaccharide as a TLR ligand in patients with primary biliary cirrhosis

Objective. Lipopolysaccharide (LPS) is suspected to trigger primary biliary cirrhosis (PBC) in susceptible individuals, yet the precise mechanism of its effect in PBC remains largely unknown. The aim of this study was to investigate altered responses to LPS ligand for Toll-like receptors (TLRs) in pathogenesis of PBC in vivo and in vitro. Material and methods. In vivo, we investigated levels of LPS and pro-inflammatory cytokines in sera and expression of LPS receptors in liver tissues from 162 patients with PBC, 325 patients with other liver diseases and 80 healthy controls. In vitro, altered responses to LPS on monocytes and cultured human biliary epithelial cells (BECs) from patients with PBC were determined. Results. Significantly higher levels of LPS in patients with PBC were detected, compared with patients with other liver diseases and healthy controls. Immunohistochemically, expression of TLR4, CD14, CD68 and NF-κB was significantly enhanced in liver tissues from patients with PBC. Before LPS stimulation, we found significantly higher serum levels of tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and IL-8 in patients with PBC than those in healthy controls. After LPS stimulation, TLR4 expression and pro-inflammatory cytokine production in CD14-positive monocytes and cultured BEC from patients with PBC increased significantly. Conclusions. These results indicated that patients with PBC were prone to exhibit higher serum LPS level, hypersensitivity of monocytes and BEC to LPS, and enhanced production of pro-inflammatory cytokines. LPS altered expression of TLR4, CD14 and NF-κB on monocytes and BEC, which may be implicated in the pathogenesis and progression of PBC.

Parenteral Nutrition Rapidly Reduces Hepatic Mononuclear Cell Numbers and Lipopolysaccharide Receptor Expression on Kupffer Cells in Mice

Parenteral nutrition (PN) reduces the number of hepatic mononuclear cell (MNCs) and impairs their function, resulting in poor survival after intraportal bacterial challenge in mice. Our recent animal study demonstrated resumption of enteral nutrition after PN to rapidly restore hepatic MNC numbers (in 12 hours) and lipopolysaccharide (LPS) receptor expression on Kupffer cells (in 48 hours). The present study examined the time courses of hepatic MNC number reductions and LPS receptor expression changes in mice receiving PN. Male mice (n = 49) from the Institute of Cancer Research were divided into chow (n = 8), PN0.5 (n = 8), PN1 (n = 8), PN2 (n = 9), PN3 (n = 9), and PN5 (n = 7) groups. The chow group was given chow with an intravenous saline infusion. The PN groups were fed parenterally for 0.5, 1, 2, 3, or 5 days following the chow-feeding courses. After 7 days of nutrition support, hepatic MNCs were isolated and counted. The expression of LPS receptors on Kupffer cells was analyzed by flow cytometry. Hepatic MNC numbers rapidly reached their lowest level in the PN0.5 and PN1 groups but were somewhat restored thereafter and remained stable after the third day, without significant differences between any 2 of the PN groups. CD14 and Toll-like receptor 4/MD-2 expressions both showed significant reductions in the PN1 group compared with the chow group and gradually decreased to their lowest levels in the PN5 group. PN administration rapidly reduces hepatic MNC numbers and LPS receptor expression on Kupffer cells.

Three-dimensional spheroid cultures of A549 and HepG2 cells exhibit different lipopolysaccharide (LPS) receptor expression and LPS-induced cytokine response compared with monolayer cultures

Lipopolysaccharide (LPS) is a potent modulator of pathogen-induced host inflammatory responses. Lipopolysaccharide signaling to host cells is correlated with the expression of well-characterized LPS receptors. We have developed three-dimensional (3-D) cell cultures (spheroids) that are more representative of in vivo conditions than traditional monolayer cultures and may provide novel in vitro models to study the inflammatory response. In this work, we have compared F-actin organization, LPS-induced pro-inflammatory cytokine response and LPS receptor expression between spheroid and monolayer cultures from A549 lung epithelial cells and HepG2 hepatocytes. Significant junctional F-actin was seen at the cell-cell contact points throughout the spheroids, while monolayer cells showed stress fibers of actin and more prominent F-actin localized at the cell base. A time course of cytokine release in response to LPS showed that A549 spheroids secreted persistently higher levels of interleukin (IL)-6 and IL-8 compared with monolayer cultures. Unlike monolayer cultures, HepG2 spheroids responded to LPS by releasing a significant level of IL-8. We identified a significant increase in the expression of CD14 and MD2 in these spheroids compared with monolayers, which may explain the enhanced cytokine response to LPS. Thus, we suggest that 3-D spheroid cell cultures are more typical of in vivo cell responses to LPS during the development of inflammation and would be a better in vitro model in inflammation studies.

A phenolic acid phenethyl urea compound inhibits lipopolysaccharide-induced production of nitric oxide and pro-inflammatory cytokines in cell culture

We previously used the Curtius rearrangement to synthesize various phenolic acid phenethyl urea compounds from phenolic acids and demonstrated their beneficial anti-oxidant and anti-cancer effects. Here, we investigated the effects of one of these synthetic compounds, ( E)-1-(3,4-dihydroxystyryl)-3-(4-hydroxyphenethyl)urea (DSHP-U), on nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression, and cytokine secretion in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. DSHP-U suppressed LPS-induced NO production and iNOS expression at a concentration of 50 μM and inhibited LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase. Inhibitors of phosphorylated (p)-ERK and p-p38, but not of p-JNK, reduced LPS-stimulated NO production. DSHP-U also prevented the nuclear translocation of the Rel A (p65) subunit and DNA-NF-κB binding by suppressing IκBα phosphorylation and by the degradation of IκBα in LPS-stimulated cells. Furthermore, DSHP-U decreased the production of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 in LPS-treated macrophages. However, the LPS-stimulated expression of LPS receptors, such as Toll-like receptor 4, myeloid differentiation factor-2, and CD14, was unchanged after DSHP-U treatment at significantly high levels. Our data suggest that DSHP-U blocks NO and inflammatory cytokine production in LPS-stimulated macrophages and that these effects are mainly mediated through the inhibition of the ERK/p38- and NF-κB signaling pathways.

Critical Protective Role for Annexin 1 Gene Expression in the Endotoxemic Murine Microcirculation

The inflammatory response is a protective process of the body to counteract xenobiotic penetration and injury, although in disease this response can become deregulated. There are endogenous biochemical pathways that operate in the host to keep inflammation under control. Here we demonstrate that the counterregulator annexin 1 (AnxA1) is critical for controlling experimental endotoxemia. Lipopolysaccharide (LPS) markedly activated the AnxA1 gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolar macrophages--cell types known to function in experimental endotoxemia. Administration of LPS to AnxA1-deficient mice produced a toxic response characterized by organ injury and lethality within 48 hours, a phenotype rescued by exogenous application of low doses of the protein. In the absence of AnxA1, LPS generated a deregulated cellular and cytokine response with a marked degree of leukocyte adhesion in the microcirculation. Analysis of LPS receptor expression in AnxA1-null macrophages indicated an aberrant expression of Toll-like receptor 4. In conclusion, this study has detailed cellular and biochemical alterations associated with AnxA1 gene deletion and highlighted the impact of this protective circuit for the correct functioning of the homeostatic response to sublethal doses of LPS.

Interleukin 8 secretion from monocytes of subjects heterozygous for the deltaF508 cystic fibrosis transmembrane conductance regulator gene mutation is altered.

Patients with cystic fibrosis (CF) exhibit an excessive host inflammatory response. The aim of this study was to determine (i) whether interleukin 8 (IL-8) secretion is increased from monocytes from subjects heterozygous as well as homozygous for cystic fibrosis transmembrane conductance regulator (CFTR) mutations and (ii) whether this is due to increased cell surface lipopolysaccharide (LPS) receptors or, alternatively, increased activation of mitogen-activated protein kinases (MAPK). The basal level of IL-8 secretion was higher from monocytes from CF patients than from monocytes from healthy controls (P = 0.02) and obligate heterozygotes (parents of the CF patients). The 50% effective concentrations for LPS-induced IL-8 production for monocytes from both CF patients and obligate heterozygotes were 100-fold lower than those for monocytes from healthy controls (P < 0.05). No differences in the levels of IL-1beta production were seen between these groups. Expression of the LPS surface receptors CD14 and Toll-like receptor 4 were not different between CF patients and healthy controls. In contrast, phosphorylation of the MAPKs p38 and ERK occurred at lower doses of LPS in monocytes from patients heterozygous and homozygous for CFTR mutations. These results indicate that a single allelic CFTR mutation is sufficient to augment IL-8 secretion in response to LPS. This is not a result of increased LPS receptor expression but, rather, is associated with alterations in MAPK signaling.

Cytosolic phospholipase A2 is responsible for prostaglandin E2 and leukotriene B4 formation in phagocyte-like PLB-985 cells: studies of differentiated cPLA2-deficient PLB-985 cells.

Our previously established model of cytosolic phospholipase A(2) (cPLA(2))-deficient, differentiated PLB-985 cells (PLB-D cells) was used to determine the physiological role of cPLA(2) in eicosanoid production. Parent PLB-985 (PLB) cells and PLB-D cells were differentiated toward the monocyte or granulocyte lineages using 5 x 10(-)(8) M 1,25 dihydroxyvitamin D(3) or 1.25% dimethyl sulfoxide, respectively. Parent monocyte- or granulocyte-like PLB cells released prostaglandin E(2) (PGE(2)) when stimulated by ionomycin, A23187, opsonized zymosan, phorbol 12-myristate 13-acetate, or formyl-Met-Leu-Phe (fMLP), and monocyte- or granulocyte-like PLB-D cells did not release PGE(2) with any of the agonists. The kinetics of cPLA(2) translocation to nuclear fractions in monocyte-like PLB cells stimulated with fMLP or ionomycin was in correlation with the kinetics of PGE(2) production. Granulocyte-like PLB cells, but not granulocyte-like PLB-D cells, secreted leukotriene B(4) (LTB(4)) after stimulation with ionomycin or A23187. Preincubation of monocyte-like parent PLB cells with 100 ng/ml lipopolysaccharide (LPS) for 16 h enhanced stimulated PGE(2) production, which is in correlation with the increased levels of cPLA(2) detected in these cells. LPS preincubation was less potent in increasing PGE(2) and LTB(4) secretion and did not affect cPLA(2) expression in granulocyte-like PLB cells, which may be a result of their lower levels of surface LPS receptor expression. LPS had no effect on monocyte- or granulocyte-like PLB-D cells. The lack of eicosanoid formation in stimulated, differentiated cPLA(2)-deficient PLB cells indicates that cPLA(2) contributes to stimulated eicosanoid formation in monocyte- and granulocyte-like PLB cells.

Gamma interferon augments the intracellular pathway for lipopolysaccharide (LPS) recognition in human intestinal epithelial cells through coordinated up-regulation of LPS uptake and expression of the intracellular Toll-like receptor 4-MD-2 complex.

Although some intestinal epithelial cell lines are known to respond to lipopolysaccharide (LPS), understanding of the relationship between LPS responsiveness and the expression of LPS receptors or factors regulating LPS responsiveness of intestinal epithelial cell lines is incomplete. In this study, we demonstrate that commonly studied human intestinal epithelial cell lines can be classified into at least three different types on the basis of LPS responsiveness, Toll-like receptor-4 (TLR4) expression, and the effects of gamma interferon (IFN-gamma) on LPS responsiveness. The first phenotype, which includes the HCT-116 and Caco-2 cell lines, is characterized by relative hyporesponsiveness to LPS and diminished expression of TLR4 protein. In these cells, IFN-gamma does not induce LPS responsiveness. The second phenotype, which includes cell line SW480, exhibits a highly LPS-responsive phenotype and surface expression of TLR4 protein even in unprimed conditions. These lines are functionally similar to cells of monocytic lineage. In the third phenotype, which includes the HT-29 and Colo205 cell lines, TLR4 protein is largely present in the cytoplasmic fraction and the cells are hyporesponsive to LPS in an unprimed condition. However, priming of these cells with IFN-gamma can induce LPS responsiveness through augmentation of LPS uptake and expression of MD-2 mRNA and intracellular TLR4 proteins. Finally, these findings suggest that the Th1 cytokine IFN-gamma modulates LPS responsiveness through several mechanisms in intestinal epithelial cells and that these cells may comprise different subpopulations with distinct roles in innate immune responses.

Genetic Deletion of the Tumor Necrosis Factor Receptor p60 or p80 Sensitizes Macrophages to Lipopolysaccharide-induced Nuclear Factor-κB, Mitogen-activated Protein Kinases, and Apoptosis

Whether deletion of tumor necrosis factor (TNF) receptor 1 or 2 affects lipopolysaccharide (LPS)-mediated signaling is not understood. In this report, we used macrophages derived from wild type (wt) mice and from mice null for the type 1 receptor (p60-/-), the type 2 receptor (p80-/-), or both (p60-/- p80-/-) to investigate the effect of these receptors on LPS-mediated activation of NF-kappaB, mitogen-activated protein kinases, and apoptosis. LPS activated NF-kappaB by 3-4-fold in wt cells but by 9-10-fold in p60-/-, p80-/-, and p60-/- p80-/- macrophages. These results correlated with the IkappaBalpha kinase activation, which is needed for NF-kappaB activation. LPS-induced cyclooxygenase-2 and inducible NO synthase proteins and NO production were maximum in p60-/- p80-/- macrophages and minimum in wt cells. LPS activated C-Jun N-terminal kinase, p38MAPK, and extracellular signal-regulated kinase in wt cells, but the levels were much higher in p60-/-, p80-/-, and p60-/- p80-/- cells. LPS-induced cytotoxicity, poly(ADP-ribose) polymerase cleavage, and annexin V staining were also highest in p60-/- p80-/- cells and lowest in wt cells. The difference in LPS signaling was unrelated to the expression of LPS receptors, CD14, or toll-like receptor 4. Overall, our studies indicate that deletion of either of the TNF receptors sensitizes the macrophages to LPS and provide evidence for cross-talk between TNF and LPS signaling.

N-formyl-methionyl-leucyl-phenylalanine (fMLP) inhibits tumour necrosis factor-alpha (TNF-alpha) production on lipopolysaccharide (LPS)-stimulated human neutrophils.

During gram-negative infections bacterial components, such as LPS and formylated peptides, exert profound physiological effects on polymorphonuclear neutrophils (PMN) resulting in increased neutrophil effector activities, including the generation of oxidative metabolites, degranulation, phagocytosis and cytokine release. There is not enough evidence about the relationships between LPS and formylated bacterial peptides in the triggering and regulation of the immune inflammatory response. In this study, we present evidence indicating that pretreatment of human PMN with a prototype formylated peptide such as fMLP results in the inhibition of TNF-alpha secretion, a key molecule that plays a central role in the pathogenesis of septic shock. This inhibitory effect of fMLP does not appear to alter the expression of LPS receptors or the transcriptional pathway of the TNF-alpha mRNA, but instead, fMLP reduces the expression of the membrane form of TNF-alpha on the PMN surface. These findings indicate that fMLP, a typical proinflammatory agent, could play, at least in determined conditions, an anti-inflammatory role.

Lipopolysaccharide-induced cytokine and receptor expression and neutrophil infiltration in the liver of osteopetrosis (op/op) mutant mice.

Mice homozygous for the osteopetrosis (op) mutation are genetically deficient in macrophage colony-stimulating factor (M-CSF/CSF-1) and are characterized by defective differentiation and function of macrophages. The aim of this study is to assess the contribution of M-CSF to lipopolysaccharide (LPS)-induced cytokine expression and neutrophil infiltration in the liver. We investigated the effects of LPS administration in M-CSF-deficient op/op mutant mice. The expression of cytokines and receptors in the liver was studied by immunohistochemistry and RT-PCR. Neutrophil infiltration in the liver was also examined. After LPS administration, cytokine production and expression of LPS receptors, such as CD14 and scavenger receptor class A (MSR-A), were induced at lower levels in op/op mice than those in littermate mice. Neutrophil infiltration in the liver of op/op mice did not differ significantly from that of littermate mice. Anti-IL-8 receptor homologue and anti-C5a receptor antibody reduced the number of infiltrating neutrophils. These findings indicate that deficient macrophage activation following LPS injection in op/op mice is associated with decreased expression of CD14 and MSR-A in the liver. Thus, M-CSF plays a critical role in LPS-induced macrophage activation but does not exert a dominant role in neutrophil infiltration in the liver.