Upregulation of MIP-2 (CXCL2) expression by 15-deoxy-Delta(12,14)-prostaglandin J(2) in mouse peritoneal macrophages

A peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), has been reported to possess anti-inflammatory activity in activated monocytes/macrophages. In this study, we investigated the effect of 15d-PGJ(2) on the lipopolysaccharide (LPS)-induced expression of chemokine mRNAs, especially macrophage inhibitory protein (MIP)-2 (CXCL2), in mouse peritoneal macrophages. The inhibitory actions of the natural PPARgamma ligands, 15d-PGJ(2) and prostaglandin A1 (PGA1), on the expression of RANTES (regulated upon activation, normal T expressed and secreted; CCL5), MIP-1beta (CCL4), MIP-1alpha (CCL3), IFN-gamma-inducible protein 10 kilodaltons (IP-10; CXCL10) and monocyte chemoattractant protein-1 (MCP-1; CCL2) mRNA in LPS-treated cells were stronger than those of the synthetic PPARgamma ligands troglitazone and ciglitazone. However, 15d-PGJ(2) enhanced the expression of LPS-induced MIP-2 (CXCL2) mRNA. A specific PPARgamma antagonist (GW9662) had no effect on the inhibitory action of 15d-PGJ(2) and PGA1 in LPS-induced chemokine mRNA expression and on the synergistic action of 15d-PGJ(2) in LPS-induced MIP-2 (CXCL2) expression. Moreover, LPS itself reduced the expression of PPARgamma. Although the synergistic effect of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) mRNA expression was remarkable, the production of MIP-2 (CXCL2) in cells treated with 15d-PGJ(2) and LPS did not increase compared to the production in cells treated with LPS alone. The synergistic action of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) mRNA expression was dependent on the activation of nuclear factor-kappaB (NF-kappaB), and 15d-PGJ(2) increased the phosphorylation of p38 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in cells stimulated with LPS. These results suggest that the synergistic effect of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) expression is PPARgamma-independent, and is mediated by the p38 and SAPK/JNK pathway in mitogen-activated protein kinase signaling pathways, which activates NF-kappaB. Our data may give more insights into the different mechanisms contrary to the anti-inflammatory effect of 15d-PGJ(2) on the expression of chemokine genes.     

Upregulation of lipopolysaccharide-induced interleukin-10 by prostaglandin A1 in mouse peritoneal macrophages

The cyclopentenone prostaglandins (cyPGs) prostaglandin A1 (PGA1) and 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) have been reported to exhibit antiinflammatory activity in activated monocytes/macrophages. However, the effects of these two cyPGs on the expression of cytokine genes may differ. In this study, we investigated the mechanism of action of PGA1 in lipopolysaccharide (LPS)-induced expression of interleukin (IL)-10 mRNA in mouse peritoneal macrophages. 15d-PGJ2 inhibited expression of LPSinduced IL-10, whereas PGA1 increased LPS-induced IL-10 expression. This synergistic effect of PGA1 on LPS-induced IL-10 expression reached a maximum as early as 2 h after simultaneous PGA1 and LPS treatment (PGA1/LPS), and did not require new protein synthesis. The synergistic effect of PGA1 was inhibited by GW9662, a specific peroxisome proliferator-activated receptor (PPAR) antagonist, and Bay-11-7082, a NF-kappaB inhibitor. The extracellular signalregulated kinases (ERK) inhibitor PD98059 increased the expression of PGA1/LPS-induced IL-10 mRNA, rather than inhibiting the IL-10 expression. Moreover, PGA1 inhibited LPS-induced ERK phosphorylation. The synergistic effect of PGA1 on LPS-induced IL-10 mRNA and protein production was inhibited by p38 inhibitor PD169316, and PGA1 increased LPS-induced p38 phosphorylation. In the case of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK), the SAPK/JNK inhibitor SP600125 did not inhibit IL-10 mRNA synthesis but inhibited the production of IL-10 protein remarkably. These results suggest that the synergistic effect of PGA1 on LPS-induced IL-10 expression is NF-kappaB-dependent and mediated by mitogen-activated protein (MAP) kinases, p38, and SAPK/ JNK signaling pathways, and also associated with the PPARgamma pathway. Our data may provide more insight into the diverse mechanisms of PGA1 effects on the expression of cytokine genes.     

Feedback control of cyclooxygenase-2 expression through PPARgamma

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostaglandins (PG), plays a key role in inflammation, tumorigenesis, development, and circulatory homeostasis. The PGD(2) metabolite 15-deoxy-Delta(12, 14) PGJ(2) (15d-PGJ(2)) was identified as a potent natural ligand for the peroxisome proliferator-activated receptor-gamma (PPARgamma). PPARgamma expressed in macrophages has been postulated as a negative regulator of inflammation and a positive regulator of differentiation into foam cell associated with atherogenesis. Here, we show that 15d-PGJ(2) suppresses the lipopolysaccharide (LPS)-induced expression of COX-2 in the macrophage-like differentiated U937 cells but not in vascular endothelial cells. PPARgamma mRNA abundantly expressed in the U937 cells, not in the endothelial cells, is down-regulated by LPS. In contrast, LPS up-regulates mRNA for the glucocorticoid receptor which ligand anti-inflammatory steroid dexamethasone (DEX) strongly suppresses the LPS-induced expression of COX-2, although both 15d-PGJ(2) and DEX suppressed COX-2 promoter activity by interfering with the NF-kappaB signaling pathway. Transfection of a PPARgamma expression vector into the endothelial cells acquires this suppressive regulation of COX-2 gene by 15d-PGJ(2) but not by DEX. A selective COX-2 inhibitor, NS-398, inhibits production of PGD(2) in the U937 cells. Taking these findings together, we propose that expression of COX-2 is regulated by a negative feedback loop mediated through PPARgamma, which makes possible a dynamic production of PG, especially in macrophages, and may be attributed to various expression patterns and physiological functions of COX-2.     

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.     

15-Deoxy-delta(12,14)-prostaglandin J(2) inhibits IL-1beta-induced IKK enzymatic activity and IkappaBalpha degradation in rat chondrocytes through a PPARgamma-independent pathway

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been shown to inhibit the effects of proinflammatory cytokines such as interleukin-1beta (IL-1beta). This cytokine plays a key role in articular pathophysiologies by inducing the production of inflammatory mediators such as nitric oxide (NO) and prostaglandin E(2) (PGE(2)). We previously demonstrated that 15d-PGJ(2) was more potent than troglitazone to counteract IL-1beta effects on chondrocytes. Here, we studied the action of 15d-PGJ(2) on intracellular targets in nuclear factor-kappaB (NF-kappaB) signalling pathway in IL-1beta treated rat chondrocytes. We found that 15d-PGJ(2) decreased inhibitor kappaBalpha (IkappaBalpha) degradation but not its phosphorylation by specifically inhibiting IkappaB kinase beta (IKKbeta), but not IKKalpha, enzymatic activity. We further evaluated the involvement of PPARgamma in the anti-inflammatory action of its ligands. In chondrocytes overexpressing functional PPARgamma protein, 15d-PGJ(2) pre-treatment inhibited inducible NO synthase and COX-2 mRNA expression, nitrite and PGE(2) production, p65 translocation and NF-kappaB activation. Troglitazone or rosiglitazone pre-treatment had no effect. 15d-PGJ(2) exhibited the same effect in chondrocytes overexpressing mutated PPARgamma protein. These results suggest that 15d-PGJ(2) exerts its anti-inflammatory effect in rat chondrocytes by a PPARgamma-independent mechanism, which can be conferred to a partial inhibition of IkappaBalpha degradation.     

Induction of heme oxygenase-1 expression in murine macrophages is essential for the anti-inflammatory effect of low dose 15-deoxy-Delta 12,14-prostaglandin J2

15-Deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2), a cyclopentenone prostaglandin, displays a potent anti-inflammatory effect at micromolar concentrations (>2 microM) through direct inhibition of nuclear factor (NF)-kappa B activation. Here we show that at submicromolar concentrations (0.1-0.5 microM) 15d-PGJ2 retains the ability to suppress the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine J774 macrophages under the conditions of a prolonged incubation (>12 h). Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of 15d-PGJ2. Inhibition of HO-1 activity or scavenging carbon monoxide (CO), a byproduct derived from heme degradation, significantly attenuated the suppressive activity of 15d-PGJ2. Furthermore, LPS-induced NF-kappa B activation assessed by the inhibitory protein of NF-kappa B(I kappa B) degradation and p50 nuclear translocation was diminished in cells subjected to prolonged treatment with the low concentration of 15d-PGJ2. Treatment of cells with the protein synthesis inhibitor, cycloheximide, or the specific p38 MAP kinase inhibitor, SB203580, blocked the induction of HO-1 and suppression of LPS-induced I kappa B degradation mediated by 15d-PGJ2. Likewise, HO inhibitor and CO scavenger were effective in abolishing the inhibitory effects of 15d-PGJ2 on NF-kappa B activation induced by LPS. The functional role of CO was further demonstrated by the use of a CO releasing molecule, tricarbonyldichlororuthenium(II) dimer, which significantly suppressed LPS-induced nuclear translocation of p50 as assessed by confocal immunofluorescence. Collectively, these data suggest that even at submicromolar concentrations 15d-PGJ2 can exert an anti-inflammatory effect in macrophages through a mechanism that involves the action of HO/CO.     

The 15-deoxy-delta12,14-prostaglandin J2 inhibits the inflammatory response in primary rat astrocytes via down-regulating multiple steps in phosphatidylinositol 3-kinase-Akt-NF-kappaB-p300 pathway independent of peroxisome proliferator-activated receptor gamma

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-12,14-PGJ2 (15d-PGJ2), have been proposed as a new class of anti-inflammatory compounds because 15d-PGJ2 was able to inhibit the induction of inflammatory response genes such as inducible NO synthase (iNOS) and TNF (TNF-alpha) in a PPAR-dependent manner in various cell types. In primary astrocytes, the anti-inflammatory effects (inhibition of TNF-alpha, IL-1beta, IL-6, and iNOS gene expression) of 15d-PGJ2 are observed to be independent of PPARgamma. Overexpression (wild-type and dominant-negative forms) of PPARgamma and its antagonist (GW9662) did not alter the 15d-PGJ2-induced inhibition of LPS/IFN-gamma-mediated iNOS and NF-kappaB activation. The 15d-PGJ2 inhibited the inflammatory response by inhibiting IkappaB kinase activity, which leads to the inhibition of degradation of IkappaB and nuclear translocation of p65, thereby regulating the NF-kappaB pathway. Moreover, 15d-PGJ2 also inhibited the LPS/IFN-gamma-induced PI3K-Akt pathway. The 15d-PGJ2 inhibited the recruitment of p300 by NF-kappaB (p65) and down-regulated the p300-mediated induction of iNOS and NF-kappaB luciferase reporter activity. Coexpression of constitutive active Akt and PI3K (p110) reversed the 15d-PGJ2-mediated inhibition of p300-induced iNOS and NF-kappaB luciferase activity. This study demonstrates that 15d-PGJ2 suppresses inflammatory response by inhibiting NF-kappaB signaling at multiple steps as well as by inhibiting the PI3K/Akt pathway independent of PPARgamma in primary astrocytes.     

15-deoxy-delta12,14-prostaglandin J2-mediated ERK signaling inhibits gram-negative bacteria-induced RelA phosphorylation and interleukin-6 gene expression in intestinal epithelial cells through modulation of protein phosphatase 2A activity

We have previously shown that non-pathogenic Gram-negative Bacteroides vulgatus induces transient RelA phosphorylation (Ser-536), NF-kappaB activity, and pro-inflammatory gene expression in native and intestinal epithelial cell (IEC) lines. We now demonstrate that 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) but not prostaglandin E(2) inhibits lipopolysaccharide (LPS) (B. vulgatus)/LPS (Escherichia coli)-induced RelA phosphorylation and interleukin-6 gene expression in the colonic epithelial cell line CMT-93. This inhibitory effect of 15d-PGJ(2) was mediated independently of LPS-induced IkappaBalpha phosphorylation/degradation and RelA nuclear translocation as well as RelA DNA binding activity. Interestingly, although B. vulgatus induced nuclear expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in native epithelium of monoassociated Fisher rats, PPARgamma-specific knock-down in CMT-93 cells using small interference RNA failed to reverse the inhibitory effects of PPARgamma agonist 15d-PGJ(2), suggesting PPARgamma-independent mechanisms. In addition, 15d-PGJ(2) but not the synthetic high affinity PPARgamma ligand rosiglitazone triggered ERK1/2 phosphorylation in IEC, and most importantly, MEK1 inhibitor PD98059 reversed the inhibitory effect of 15dPGJ(2) on LPS-induced RelA phosphorylation and interleukin-6 gene expression. Calyculin A, a specific phosphoserine/phospho-threonine phosphatase inhibitor increased the basal phosphorylation of RelA and reversed the inhibitory effect of 15d-PGJ(2) on LPS-induced RelA phosphorylation. We further demonstrated in co-immunoprecipitation experiments that 15d-PGJ(2) triggered protein phosphatase 2A activity, which directly dephosphorylated RelA in LPS-stimulated CMT-93 cells. We concluded that 15d-PGJ(2) may help to control NF-kappaB signaling and normal intestinal homeostasis to the enteric microflora by modulating RelA phosphorylation in IEC through altered protein phosphatase 2A activity.     

A novel chromone derivative with anti-inflammatory property via inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway

The p38 MAPK signaling pathway plays a pivotal role in inflammation. Targeting p38 MAPK may be a potential strategy for the treatment of inflammatory diseases. In the present study, we show that a novel chromone derivative, DCO-6, significantly reduced lipopolysaccharide (LPS)-induced production of nitric oxide, IL-1β and IL-6, decreased the levels of iNOS, IL-1β and IL-6 mRNA expression in both RAW264.7 cells and mouse primary peritoneal macrophages, and inhibited LPS-induced activation of p38 MAPK but not of JNK, ERK. Moreover, DCO-6 specifically inhibited TLR4-dependent p38 activation without directly inhibiting its kinase activity. LPS-induced production of intracellular reactive oxygen species (ROS) was remarkably impaired by DCO-6, which disrupted the formation of the TRAF6-ASK1 complex. Administering DCO-6 significantly protected mice from LPS-induced septic shock in parallel with the inhibition of p38 activation and ROS production. Our results indicate that DCO-6 showed anti-inflammatory properties through inhibition of ROS-dependent activation of TRAF6-ASK1-p38 pathway. Blockade of the upstream events required for p38 MAPK action by DCO-6 may provide a new therapeutic option in the treatment of inflammatory diseases.     

Differential regulation of chemokine gene expression by 15-deoxy-delta 12,14 prostaglandin J2

Ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as 15-deoxy-Delta(12,14)PGJ2 (15d-PGJ2) have been proposed as a new class of antiinflammatory compounds with possible clinical applications. As there is some controversy over the inhibitory effects of 15d-PGJ2 on chemokine gene expression, we investigated whether 15d-PGJ2 itself affected chemokine gene expression in human monocytes/macrophages and two monocytic cell lines. Here we demonstrate that the 15d-PGJ2 can induce IL-8 gene expression. In contrast, monocyte chemoattractant protein-1 gene expression was suppressed by 15d-PGJ2, while the expression of RANTES was unaltered. Furthermore, concomitant treatment of monocytes/macrophages with 15d-PGJ2 (2.5 x 10(-6) M) potentiated LPS-induced gene expression of IL-8 mRNA, but suppressed PMA-induction of IL-8 mRNA. In addition, treatment of U937 and THP-1 cells with 15d-PGJ2 also resulted in induction of IL-8 gene expression. Further studies demonstrated that 15d-PGJ2 regulated IL-8 gene expression via a ligand-specific and PPARgamma-dependent pathway. Our observations revealed a previous unappreciated function and mechanism of 15d-PGJ2-mediated regulation of cytokine gene expression in monocytes/macrophages.     

15-deoxy-Delta12,14-prostaglandin J2 inhibits IFN-inducible protein 10/CXC chemokine ligand 10 expression in human microglia: mechanisms and implications

Regulation of cytokine and chemokine expression in microglia may have implications for CNS inflammatory disorders. In this study we examined the role of the cyclopentenone PG 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in microglial inflammatory activation in primary cultures of human fetal microglia. 15d-PGJ(2) potently inhibited the expression of microglial cytokines (IL-1, TNF-alpha, and IL-6). We found that 15d-PGJ(2) had differential effects on the expression of two alpha-chemokines; whereas the Glu-Lys-Arg (ELR)(-) chemokine IFN-inducible protein-10/CXCL10 was inhibited, the ELR(+) chemokine IL-8/CXCL8 was not inhibited. These findings were shown in primary human microglia and the human monocytic cells line THP-1 cells, using diverse cell stimuli such as bacterial endotoxin, proinflammatory cytokines (IL-1 and TNF-alpha), IFN-beta, and HIV-1. Furthermore, IL-8/CXCL8 expression was induced by 15d-PGJ(2) alone or in combination with TNF-alpha or HIV-1. Combined results from EMSA, Western blot analysis, and immunocytochemistry showed that 15d-PGJ(2) inhibited NF-kappaB, Stat1, and p38 MAPK activation in microglia. Adenoviral transduction of super-repressor IkappaBalpha, dominant negative MKK6, and dominant negative Ras demonstrated that NF-kappaB and p38 MAPK were involved in LPS-induced IFN-inducible protein 10/CXCL10 production. Interestingly, although LPS-induced IL-8/CXCL8 was dependent on NF-kappaB, the baseline or 15d-PGJ(2)-mediated IL-8/CXCL8 production was NF-kappaB independent. Our results demonstrate that 15d-PGJ(2) has opposing effects on the expression of two alpha-chemokines. These data may have implications for CNS inflammatory diseases.     

Proteinase-activated receptor 2 activation promotes an anti-inflammatory and alternatively activated phenotype in LPS-stimulated murine macrophages

Proteinase-activated receptor 2 (PAR(2)), a 7-transmembrane G protein-coupled receptor, contributes to inflammation either positively or negatively in different experimental systems. Previously, we reported that concurrent activation of PAR(2) and TLRs in human lung and colonic epithelial cells resulted in a synergistic increase in NF-κB-mediated gene expression, but a down-regulation of IRF-3-mediated gene expression. In this study, the effect of PAR(2) activation on LPS-induced TLR4 signaling was examined in primary murine macrophages. The PAR(2) activation of wild-type macrophages enhanced LPS-induced expression of the anti-inflammatory cytokine, IL-10, while suppressing gene expression of pro-inflammatory cytokines, TNF-α, IL-6, and IL-12. Similar PAR(2)-mediated effects on LPS-stimulated IL-10 and IL-12 mRNA were also observed in vivo. In contrast, PAR 2-/- macrophages exhibited diminished LPS-induced IL-10 mRNA and protein expression and downstream STAT3 activation, but increased KC mRNA and protein. PAR(2) activation also enhanced both rIL-4- and LPS-induced secretion of IL-4 and IL-13, and mRNA expression of alternatively activated macrophage (AA-M) markers, e.g. arginase-1, mannose receptor, Ym-1. Thus, in the context of a potent inflammatory stimulus like LPS, PAR(2) activation acts to re-establish tissue homeostasis by dampening the production of inflammatory mediators and causing the differentiation of macrophages that may contribute to the development of a Th2 response.     

The synergistic anti-inflammatory effects of lycopene, lutein, β-carotene, and carnosic acid combinations via redox-based inhibition of NF-κB signaling

Inflammatory mediators and cytokines play important roles in the pathogenesis of a vast number of human diseases; therefore much attention is focused on blunting their proinflammatory modes of action. The aims of the present research were to assess the effectiveness of combinations of carotenoids and phenolics, at concentrations that can be achieved in blood, to inhibit the release of inflammatory mediators from macrophages exposed to lipopolysaccharide (LPS) and to determine what the anti-inflammatory effect of the phytonutrient combinations was in an in vivo mouse model of peritonitis. Preincubation of mouse peritoneal macrophages with lycopene (1μM) or Lyc-O-Mato (1μM) and carnosic acid (2μM), lutein (1μM), and/or β-carotene (2μM) 1h before addition of LPS for 24h caused a synergistic inhibition of NO, prostaglandin E(2), and superoxide production derived from downregulation of iNOS, COX-2, and NADPH oxidase protein and mRNA expression and synergistic inhibition of TNFα secretion. We surmise that the anti-inflammatory action of the phytonutrient combinations used probably resides in their antioxidant properties, because they caused an immediate, efficient, and synergistic inhibition of LPS-induced internal superoxide production leading to a marked decrease in ERK and NF-κB activation. The anti-inflammatory effects of the selected phytonutrient combinations were also demonstrated in a mouse model of peritonitis: their supplementation in drinking water resulted in attenuation of neutrophil recruitment to the peritoneal cavity and in inhibition of inflammatory mediator production by peritoneal neutrophils and macrophages.     

Suppression of cytokine production in lipopolysaccharide-stimulated mouse macrophages by novel cationic glucosamine derivative involves down-regulation of NF-kappaB and MAPK expressions

Exposure of macrophages to bacterial lipopolysaccharide (LPS) induces release of proinflammatory cytokines that play crucial roles in chronic inflammation. Glucosamine has reported to possess anti-inflammatory properties and currently is the oral supplement of choice for the management of inflammation related complications including osteoarthritis. In this study, quaternized amino glucosamine (QAGlc), a newly synthesized cationic glucosamine (Glc) derivative was found to inhibit LPS-stimulated production of IL-1beta, IL-6, TNF-alpha, and PGE(2) in RAW264.7, mouse macrophages more potently than its starting material Glc. Since production of cytokines is regulated mainly via activation of NF-kappaB and regulation of mitogen-activated protein kinases (MAPKs), we examined if QAGlc could be responsible for the suppression of NF-kappaB pathway and MAPKs. We used reporter gene assay and Western blotting to examine the effects of QAGlc on activation and translocation of NF-kappaB. Further, QAGlc-mediated inhibition of NF-kappaB was accompanied with a suppression of its translocation. Apparently, QAGlc was shown to attenuate LPS-induced activation of p38 MAPK and JNK in RAW264.7 cells suggesting that inhibition of MAPK-mediated LPS signaling also contribute to suppression of cytokine production following stimulation of macrophages with LPS.     

Lipopolysaccharide-induced expression of the competence gene KC in vascular endothelial cells is mediated through protein kinase C

The KC gene is a cell cycle-dependent competence gene originally identified in platelet-derived growth factor-stimulated BALB/c-3T3 cells. This gene is also induced in murine peritoneal macrophages in response to activation stimuli. We have examined the expression of the KC gene in cultured porcine aortic endothelial cells following treatment with bacterial lipopolysaccharide (LPS) as a first step in defining the early molecular events involved in endothelial cell stimulation by physiologically relevant modulators. LPS markedly elevated the steady-state level of KC mRNA in confluent endothelial cells; maximum induction of KC occurred in the cells following exposure to 10 ng/ml LPS for 2 h. LPS did not increase the growth fraction of the cells, nor was the KC mRNA level changed in dense endothelial cells stimulated to enter the cell cycle with epidermal growth factor. However, KC mRNA expression was elevated by addition of serum to starved, subconfluent endothelial cell cultures. Treatment of endothelial cells with phorbol myristate acetate (PMA) and 1-oleoyl-2-acetyl-glycerol (OAG) also induced KC gene expression. A maximum response was obtained with 10 nM PMA, the effect decreasing with higher levels of the phorbol ester. The calcium ionophore A23187 exhibited little stimulatory activity alone; however, the ionophore did cause a doubling in the PMA-stimulated KC expression. The increased expression of KC induced by LPS and PMA was inhibited by the presence of 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H7), a protein kinase C inhibitor, but not by HA1004 (an H7 analogue with little protein kinase C inhibitory activity). No cytotoxicity was observed in inhibitor or LPS-treated endothelial cell cultures. These results demonstrate that KC gene expression is stimulated by LPS in vascular endothelial cells in a proliferation-independent process. Second, unlike LPS-induced KC expression in macrophages and platelet-derived growth factor-induced KC expression in 3T3 cells, LPS induction of KC in endothelial cells appears to require activation of protein kinase C.     

Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages

The human MD-2 molecule is associated with the extracellular domain of human Toll-like receptor 4 (TLR4) and greatly enhances its LPS signaling. The human TLR4-MD-2 complex thus signals the presence of LPS. Little is known, however, about cell surface expression and LPS signaling of the TLR4-MD-2 complex in vivo. We cloned mouse MD-2 molecularly and established a unique mAb MTS510, which reacted selectively with mouse TLR4-MD-2 but not with TLR4 alone in flow cytometry. Mouse MD-2 expression in TLR4-expressing cells enhanced LPS-induced NF-kappaB activation, which was clearly inhibited by MTS510. Thioglycolate-elicited peritoneal macrophages expressed TLR4-MD-2, which was rapidly down-regulated in the presence of LPS. Moreover, LPS-induced TNF-alpha production by peritoneal macrophages was inhibited by MTS510. Collectively, the TLR4-MD-2 complex is expressed on macrophages in vivo and senses and signals the presence of LPS.