An anti-endotoxin peptide that generates from the amino-terminal domain of complement regulatory protein C1 inhibitor

C1 inhibitor (C1INH), a complement regulatory protein, prevents endotoxin shock via a direct interaction of the amino-terminal domain with gram-negative bacterial lipopolysaccharide (LPS). Importantly, the cleaved, inactive C1INH still is an anti-endotoxin effector indicating the anti-endotoxin peptide that generates from the amino-terminal domain of C1INH. In this study, we first identified that a cleaved fragment within the major part of the amino-terminal domain in in vitro proteolytic analysis of C1INH had an ability to bind to LPS. We synthesized several peptides overlapping the C1INH cleaved fragment. Among these synthetic peptides, a 13-mer derivative peptide at position from 18 to 30, named N2((18-30)), exhibited the most powerful anti-endotoxin activity in vitro, enlightening that it was most strong at binding to LPS, inhibiting the interaction of LPS with LPS-binding protein (LBP), blocking LPS binding to CD14(+) cells, and suppressing production of tumor necrosis factor (TNF)-alpha by murine macrophages, RAW 264.7. In the murine endotoxin shock model, the peptide N2((18-30)) protected mice from LPS-induced lethal septic shock by inhibiting macrophage activation. These data indicate that the peptide N2((18-30)) derived from the amino-terminal region of C1INH is anti-endotoxin.     

Cathelicidin family of antibacterial peptides CAP18 and CAP11 inhibit the expression of TNF-alpha by blocking the binding of LPS to CD14(+) cells.

Mammalian myeloid and epithelial cells express several kinds of antibacterial peptides (alpha-/beta-defensins and cathelicidins) that contribute to the innate host defense by killing invading micro-organisms. In this study we evaluated the LPS-neutralizing activities of cathelicidin peptides human CAP18 (cationic antibacterial proteins of 18 kDa) and guinea pig CAP11 using the CD14(+) murine macrophage cell line RAW264.7 and the murine endotoxin shock model. Flow cytometric analysis revealed that CAP18 and CAP11 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, Northern and Western blot analyses indicated that CAP18 and CAP11 suppressed LPS-induced TNF-alpha mRNA and protein expression by RAW264.7 cells. Interestingly, CAP18 and CAP11 possessed LPS-binding activities, and they strongly suppressed the interaction of LPS with LPS binding protein that mediates the transport of LPS to CD14 to facilitate the activation of CD14(+) cells by LPS. Moreover, when CAP18 and CAP11 were preincubated with RAW264.7 cells, they bound to the cell surface CD14 and inhibited the binding of FITC-LPS to the cells. Furthermore, in the murine endotoxin shock model, CAP18 or CAP11 administration inhibited the binding of LPS to CD14(+) cells (peritoneal macrophages) and suppressed LPS-induced TNF-alpha expression by these cells. Together these observations indicate that cathelicidin peptides CAP18 and CAP11 probably exert protective actions against endotoxin shock by blocking the binding of LPS to CD14(+) cells, thereby suppressing the production of cytokines by these cells via their potent binding activities for LPS and CD14.     

Identification of a novel inhibitor of LPS-induced TNF-alpha production with antiproliferative activity in monocyte/macrophages

An isoquinoline derivative, 5-methyl-7,8-dimethoxy-1-phenylpyrazolo[5,4-c]isoquinoline (compound 1), was identified as a novel inhibitor of LPS-induced TNF-alpha production by cell-based screening. Compound 1 suppressed LPS-induced TNF-alpha production in RAW264.7 cells and murine peritoneal macrophages in a dose-dependent manner similar to SB203580, known as a specific inhibitor of p38 MAPK. It also inhibited an LPS-induced increase in serum TNF-alpha in a mouse endotoxic shock model with an ED(50) of approximately 10 mg/kg. Compound 1 had little effect on the incorporation of [3H]-leucine into the cells, while it suppressed LPS-induced TNF-alpha mRNA levels in RAW264.7 cells. The results indicate that suppression of TNF-alpha production was not a result of nonspecific inhibition of de novo translation but was based on the decreased TNF-alpha mRNA levels. The in vitro kinase assay revealed that compound 1 did not strongly inhibit p38 MAPK activity, its potency being much lower than that of SB203580, suggesting that the TNF-alpha-suppressive action of compound 1 cannot be attributed to the inhibition of p38 MAPK. Furthermore, in contrast to SB203580, it significantly inhibited the growth of RAW264.7 and THP-1 cells in a cytostatic manner. Compound 1 is likely to have antiinflammatory and antiproliferative effects by acting on some molecule other than p38 MAPK that contributes to both LPS-induced TNF-alpha production and the cell growth of monocyte/macrophages.     

Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-alpha in macrophage cell line and rat Kupffer cells

Recent studies have demonstrated that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis. However, the mechanism responsible for the beneficial effect of AM/AMBP-1 remains unknown. The aim of this study therefore was to determine whether AM/AMBP-1 directly reduces lipopolysaccharide (LPS)-induced secretion of TNF-alpha from murine macrophage-like cell line RAW 264.7 cells and Kupffer cells isolated from normal rats. TNF-alpha release and gene expression were determined by ELISA and RT-PCR, respectively. The results indicated that LPS increased TNF-alpha production from RAW cells by 38-63-fold in a dose- and time-dependent manner. Although incubation with AM or AMBP-1 alone inhibited LPS-induced TNF-alpha release by 14-22% and 13-22%, respectively, AM and AMBP-1 in combination significantly suppressed TNF-alpha production (by 24-35%). Moreover, the upregulated TNF-alpha mRNA by LPS stimulation was significantly reduced by AM/AMBP-1, but not by AM or AMBP-1 alone. In the Kupffer cells primary culture, AM or AMBP-1 alone inhibited LPS-induced TNF-alpha production by 52% and 44%, respectively. Co-culture with AM/AMBP-1 markedly reduced TNF-alpha production (by 90%). Moreover, AM or AMBP-1 alone decreased TNF-alpha mRNA expression by 41% and 36%, respectively, whereas the combination of AM/AMBP-1 decreased its expression by 63%. These results indicate that AM and AMBP-1 in combination effectively suppress LPS-induced TNF-alpha expression and release especially from primary cultured Kupffer cells, suggesting that the downregulatory effect of AM/AMBP-1 on proinflammatory cytokine TNF-alpha may represent a mechanism responsible for their beneficial effects in preventing inflammatory responses and tissue damage in sepsis.     

5-Aminoimidazole-4-carboxamide riboside suppresses lipopolysaccharide-induced TNF-alpha production through inhibition of phosphatidylinositol 3-kinase/Akt activation in RAW 264.7 murine macrophages

5-Aminoimidazole-4-carboxamide riboside (AICAR) is an adenosine analog and a widely used activator of AMP-activated protein kinase (AMPK). We examined the effect of AICAR on LPS-induced TNF-alpha production in RAW 264.7 and peritoneal macrophages and its molecular mechanism in RAW 264.7 macrophages. Treatment with AICAR inhibited LPS-induced increases in TNF-alpha mRNA and protein levels in these cells. AICAR or LPS did not alter the AMPK activity as well as the phosphorylations of AMPK alpha (Thr172) and ACC (Ser79). Moreover, an adenosine kinase inhibitor 5'-iodotubercidin enhanced the suppressive effect of AICAR on TNF-alpha levels. These results suggest that the effect of AICAR on TNF-alpha suppression in RAW 264.7 cells is independent of AMPK activation. In addition, an adenosine receptor antagonist 8-SPT had no effect on AICAR-induced suppression of TNF-alpha levels. Finally, we observed that AICAR inhibited LPS-induced activation of PI 3-kinase and Akt, whereas it had no effect on the activation of p38 and ERK1/2. Taken together, these results suggest that the anti-inflammatory action of AICAR in RAW 264.7 macrophages is independent of AMPK activation and is associated with inhibition of LPS-induced activation of PI 3-kinase/Akt pathway.     

Heat shock inhibits IL-12 p40 expression through NF-kappa B signalling pathway in murine macrophages.

We report the effect of heat shock on lipopolysaccharide (LPS)-induced interleukin 12 (IL-12) expression. The augmentation of LPS-induced IL-12 p40 mRNA and p70 protein was significantly suppressed in both peritoneal macrophages and RAW264.7 cells after heat shock at 43 degrees C. The binding activity of nuclear factor kappa B (NF-kappa B) was reduced by prior heat shock. LPS did not induce degradation of the inhibitory protein I-kappa B alpha in the shocked cells, which might be a potential mechanism to block NF-kappa B activation. Furthermore, transient transfection assay in RAW264.7 cells demonstrated that LPS-induced activation of DM703 and DM138 (contains NF-kappa B motif) was highly sensitive to heat shock. These data suggest that heat shock influences expression of IL-12 through the I-kappa B/NF-kappa B pathway.     

A role of mitogen and stress-activated protein kinase 1/2 in survival of lipopolysaccharide-stimulated RAW 264.7 macrophages

The effect of inhibition of mitogen and stress-activated protein kinases 1/2 (MSK1/2) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was investigated. Pretreatment with Ro 31-8220, an inhibitor of MSK1/2, induced cell death in LPS-stimulated RAW 264.7 cells. In contrast, calphostin C, another inhibitor of protein kinase C, did not cause cell death. Cell death was not mediated by the release of pro-inflammatory mediators from LPS-stimulated RAW 264.7 cells. Cell death was accompanied by DNA fragmentation and annexin V binding, suggesting apoptotic cell death. Further, several caspase inhibitors did not prevent LPS-induced cell death of Ro 31-8220-pretreated RAW 264.7 cells. Nuclear translocation of apoptosis-inducing factor (AIF) was detected in Ro 31-8220-pretreated cells after LPS stimulation. Cell death was due to mitochondrial damage. Ro 31-8220 exclusively inhibited the phosphorylation of cAMP-responsive element binding protein (CREB), a substrate of MSK1/2. RAW 264.7 cells transfected with the dominant-negative MSK1 clones underwent cell death in response to LPS. Hence, it was suggested that MSK1/2 might play a critical role in the survival of LPS-stimulated RAW 264.7 cells.     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

C-Mannosylated peptides derived from the thrombospondin type 1 repeat enhance lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells

C-Mannosylation is a unique type of glycosylation occurring at the first Trp (W) in the WXXW motif, which is found in the thrombospondin type 1 repeat (TSR) of proteins. However, the biological function of C-mannosylation is not fully understood. In this study, we investigated the effect of C-mannosylated TSR-derived peptides on lipopolysaccharide (LPS)-induced signaling in macrophage-like RAW264.7 cells. The cells were stimulated with LPS in the presence or absence of chemically synthesized peptides with or without C-mannose (e.g., (C-Man)-Trp-Ser-Pro-Trp [C-Man-WSPW], C-Man-W, WSPW, etc.), then the effects of the peptides on cellular viability and signaling were examined. We found a cytotoxic effect in the cells treated with LPS and C-Man-WSPW, but not in the cells solely treated with LPS or C-Man-WSPW. We also found that production of tumor necrosis factor-alpha (TNF-alpha) was upregulated more in response to LPS plus C-Man-WSPW, than in response to LPS plus WSPW or LPS alone. Among the LPS-induced signaling pathways that induce production of TNF-alpha, the activation of c-Jun N-terminal kinase (JNK) was greatly enhanced by LPS and C-Man-WSPW, and the production of TNF-alpha was suppressed by an inhibitor for JNK. Together, these results demonstrate a novel function of the C-mannosylated TSR-derived peptide motif, to promote LPS-induced JNK signaling, and this leads to an enhancement of cytotoxicity via the upregulation of TNF-alpha production.     

Lipopolysaccharide stimulation of ERK1/2 increases TNF-alpha production via Egr-1

Lipopolysaccharide (LPS) is a potent activator of tumor necrosis factor-alpha (TNF-alpha) production by macrophages. LPS stimulates the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and increases TNF-alpha mRNA and protein accumulation in RAW 264.7 murine macrophages. However, the role of ERK1/2 activation in mediating LPS-stimulated TNF-alpha production is not well understood. Inhibition of ERK1/2 activation with PD-98059 or overexpression of dominant negative ERK1/2 decreased LPS-induced TNF-alpha mRNA quantity. LPS rapidly increased early growth response factor (Egr)-1 binding to the TNF-alpha promoter; this response was blunted in cells treated with PD-98059 or transfected with dominant-negative ERK1/2. Using a chloramphenicol acetyltransferase reporter gene linked to the Egr-1 promoter, we show that LPS increased Egr-1 promoter activity via an ERK1/2-dependent mechanism. These results delineate the role of ERK1/2 activation of Egr-1 activity in mediating LPS-induced increases in TNF-alpha mRNA expression in macrophages.     

Interleukin-10 inhibits tumor necrosis factor-alpha production in lipopolysaccharide-stimulated RAW 264.7 cells through reduced MyD88 expression

The mechanism of interleukin (IL)-10-mediated inhibition of tumor necrosis factor (TNF)-alpha production was studied by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. IL-10 inhibited TNF-alpha production transiently at an early stage after LPS stimulation. IL-10 inhibited the activation of nuclear factor (NF)-kappaB, p38 and stress-activated protein kinase (SAPK) in LPS-stimulated RAW 264.7 cells. Although the level of MyD88 protein increased in response to LPS, IL-10 prevented the LPS-induced MyD88 augmentation. There was no significant difference in the MyD88 mRNA expression between the cells pretreated with or without IL-10 in response to LPS. Therefore, IL-10 was suggested to inhibit LPS-induced TNF-alpha production via reduced MyD88 expression.     

Hypoxia modulates lipopolysaccharide induced TNF-alpha expression in murine macrophages

The pro-inflammatory activity of Tumor necrosis factor-alpha (TNF-alpha) together with tissue hypoxia determine the clinical outcome in sepsis and septic shock. p38 MAPKinase is the primary intracellular signaling pathway that regulates lipopolysaccharide (LPS)-induced TNF-alpha biosynthesis, however, the effect of hypoxia on LPS mediated activation of p38 is not known. Here we report that SB203580, a specific p38 MAPK inhibitor, which completely abolished LPS-induced TNF-alpha expression by the mouse macrophage cell RAW264.7 in normoxic conditions, lost the inhibitory effect in hypoxic conditions. Hypoxia did not modulate expression of p38 MAPK, but increased that of p-MK2, a downstream target of p38 MAPK. In LPS induced endotoxemia mice model SB203580 had no inhibitory effect on the serum levels of TNF-alpha. Furthermore, hypoxia inducible factor-1alpha (HIF-1alpha) was detected in vivo after LPS administration but its expression was not affected by SB203580. Our data indicate that LPS induced p38 MAPK activation was enhanced by hypoxia and consequently increased TNF-alpha secretion. Furthermore, the induction of HIF-1alpha in mice with endotoxemia suggested a synergistic effect on p38 mediated TNF-alpha expression. These findings provide new insights on the pathophysiological effects of hypoxia in sepsis and septic shock.     

Short-term treatment of RAW264.7 macrophages with adiponectin increases tumor necrosis factor-alpha (TNF-alpha) expression via ERK1/2 activation and Egr-1 expression: role of TNF-alpha in adiponectin-stimulated interleukin-10 production

Adiponectin is an adipokine with potent anti-inflammatory properties. However, the mechanisms by which adiponectin suppresses macrophage function are not well understood. Treatment of RAW264.7 macrophages with adiponectin for 18 h decreased lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) production. Here we demonstrate that globular adiponectin (gAcrp) initially increased TNF-alpha expression in RAW264.7 macrophages; this TNF-alpha then contributed to increased expression of interleukin-10, which in turn was required for the development of tolerance to subsequent LPS exposure. gAcrp-mediated increases in TNF-alpha mRNA accumulation were associated with increased TNF-alpha promoter activity. gAcrp increased the DNA binding activity of both Egr-1 and NFkappaB; mutation of either the Egr-1 or NFkappaB binding sites in the TNF-alpha promoter decreased gAcrp-stimulated promoter activity. Further, co-transfection with either dominant negative Egr-1 or the IkappaB super-repressor prevented gAcrp-stimulated TNF-alpha promoter activity. gAcrp also increased Egr-1 promoter activity, mRNA accumulation, and DNA binding activity. Inhibition of ERK1/2 with U0126 potently suppressed gAcrp-stimulated Egr-1 promoter activity, as well as TNF-alpha promoter activity. In summary, these data demonstrate that adiponectin initially increases TNF-alpha production by macrophages via ERK1/2-->Egr-1 and NFkappaB-dependent mechanisms; these increases in TNF-alpha in turn lead to increased expression of interleukin-10 and an eventual dampening of LPS-mediated cytokine production in macrophages.     

Lipopolysaccharide induces hyporesponsiveness to its own action in RAW 264.7 cells

Bacterial endotoxin (lipopolysaccharide, LPS) has the property of inducing hyporesponsiveness or tolerance to its own effects. This phenomenon has been demonstrated in man and experimental animals. The cellular changes that contribute to LPS tolerance are not understood. One mechanism of tolerance could involve a diminished response to LPS by key effector cells such as macrophages. Here we describe experiments designed to determine the mechanism whereby LPS produces a hyporesponsive state to its own effects. Because of the importance of the monokine known as tumor necrosis factor-alpha (TNF-alpha) in mediating many of the diverse effects of LPS, we have studied induction of TNF-alpha at the mRNA and activity level in the murine macrophage-like cell line RAW 264.7. Hyporesponsiveness can be induced by exposure of RAW 264.7 cells to low doses of LPS for more than 6 h prior to challenge with a second, normally stimulatory dose of LPS. This hyporesponsiveness is characterized by a diminished ability of LPS to increase steady state levels of TNF-alpha mRNA, is not due to an increased rate of TNF-alpha mRNA degradation, and is specific for LPS since LPS-pretreated and control cells produce similar amounts of TNF-alpha in response to challenge with heat-killed Staphylococcal aureus. The presence of indomethacin during the primary and/or challenge LPS treatment has no effect on the induction of acquired hyporesponsiveness. Thus, cyclooxygenase products are probably not involved in the development of LPS-induced hyporesponsiveness. These studies provide the basis for a better understanding of the cellular mechanisms that contribute to LPS tolerance.     

Bacterial lipopolysaccharide induces osteoclast formation in RAW 264.7 macrophage cells

Lipopolysaccharide (LPS) is a potent bone resorbing factor. The effect of LPS on osteoclast formation was examined by using murine RAW 264.7 macrophage cells. LPS-induced the formation of multinucleated giant cells (MGC) in RAW 264.7 cells 3 days after the exposure. MGCs were positive for tartrate-resistant acid phosphatase (TRAP) activity. Further, MGC formed resorption pits on calcium-phosphate thin film that is a substrate for osteoclasts. Therefore, LPS was suggested to induce osteoclast formation in RAW 264.7 cells. LPS-induced osteoclast formation was abolished by anti-tumor necrosis factor (TNF)-alpha antibody, but not antibodies to macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL). TNF-alpha might play a critical role in LPS-induced osteoclast formation in RAW 264.7 cells. Inhibitors of NF-kappaB and stress activated protein kinase (SAPK/JNK) prevented the LPS-induced osteoclast formation. The detailed mechanism of LPS-induced osteoclast formation is discussed.