Antibacterial cathelicidin peptide CAP11 suppresses the anandamide production from lipopolysaccharide-stimulated mononuclear phagocytes

The action of antibacterial cathelicidin peptide CAP11 on the anandamide production from mononuclear phagocytes was examined. Lipopolysaccharide (LPS)-stimulation induced the anandamide production from macrophage-like RAW264.7, accompanied with the enhanced anandamide-synthesizing enzyme activity; however, the anandamide-degrading enzyme activity was not changed by LPS-stimulation. Importantly, CAP11 suppressed the LPS-induced anandamide production and the increase of anandamide-synthesizing enzyme activity. Furthermore, CAP11 abrogated the LPS-binding to CD14-positive RAW264.7. These observations indicate that CAP11 inhibits the binding of LPS to CD14-positive mononuclear phagocytes, thereby suppressing the anandamide synthesizing enzyme activity and the anandamide production from the cells.     

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.     

Cutting edge: cationic antimicrobial peptides block the binding of lipopolysaccharide (LPS) to LPS binding protein

We investigated the mechanism by which cationic antimicrobial peptides block the activation of macrophages by LPS. The initial step in LPS signaling is the transfer of LPS to CD14 by LPS binding protein (LBP). Because many cationic antimicrobial peptides bind LPS, we asked whether these peptides block the binding of LPS to LBP. Using an assay that measures the binding of LPS to immobilized LBP, we show for the first time that a variety of structurally diverse cationic antimicrobial peptides block the interaction of LPS with LBP. The relative ability of different cationic peptides to block the binding of LPS to LBP correlated with their ability to block LPS-induced TNF-alpha production by the RAW 264.7 macrophage cell line.     

C1 inhibitor prevents endotoxin shock via a direct interaction with lipopolysaccharide

C1 inhibitor (C1INH) is beneficial in animal models of endotoxemia and sepsis. However, the mechanism(s) of C1INH protection remain(s) ill-defined. In this study, we demonstrated that both active C1INH and reactive center-cleaved, inactive C1INH protected mice from lethal Gram-negative endotoxemia. Both forms of C1INH blocked the LPS-binding protein-dependent binding of Salmonella typhimurium LPS to the murine macrophage cell line, RAW 264.7, and suppressed LPS-induced TNF-alpha mRNA expression. Inhibition of LPS binding to RAW 264.7 cells was reversed with anti-C1INH Ab and was more efficient when C1INH was incubated first with LPS rather than with the cells. C1INH also suppressed LPS-induced up-regulation of TNF-alpha mRNA in whole human blood. The interaction of C1INH with LPS was directly demonstrated both by ELISA and by nondenaturing PAGE, but deletion of the amino-terminal 97-aa residues abrogated this binding. Therefore, C1INH, in addition to its function as a serine protease inhibitor, has a novel anti-inflammatory function mediated via its heavily glycosylated amino-terminal non-serpin domain.     

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.     

Ailanthoidol suppresses lipopolysaccharide-stimulated inflammatory reactions in RAW264.7 cells and endotoxin shock in mice

The biological properties of ailanthoidol, a neolignan from Zanthoxylum ailanthoides or Salvia miltiorrhiza Bunge, which is used in Chinese traditional herbal medicine, have not been evaluated. Here, we report that ailanthoidol inhibits inflammatory reactions in macrophages and protects mice from endotoxin shock. Our in vitro experiments showed that ailanthoidol suppressed the generation of nitric oxide (NO) and prostaglandin E(2) , as well as the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 induced by lipopolysaccharide (LPS) in RAW264.7 cells. Similarly, ailanthoidol inhibited the production of inflammatory cytokines induced by LPS in RAW264.7 cells, including interleukin (IL)-1β and IL-6. In an animal model, ailanthoidol protected BALB/c mice from LPS-induced endotoxin shock, possibly through inhibition of the production of inflammatory cytokines and NO. Collectively, ailanthoidol inhibited the production of inflammatory mediators and may be a potential target for treatment of various inflammatory diseases.     

AS-703026 Inhibits LPS-Induced TNFα Production through MEK/ERK Dependent and Independent Mechanisms

Chronic obstructive pulmonary disease (COPD) is characterized by intense lung infiltrations of immune cells (macrophages and monocytes). Lipopolysaccharide (LPS) activates macrophages/monocytes, leading to production of tumor necrosis factor α (TNFα) and other cytokines, which cause subsequent lung damages. In the current study, our results demonstrated that AS-703026, a novel MEK/ERK inhibitor, suppressed LPS-induced TNFα mRNA expression and protein secretion in RAW 264.7 murine macrophages, and in murine bone marrow-derived macrophages (BMDMs). Meanwhile, TNFα production in LPS-stimulated COPD patents’ peripheral blood mononuclear cells (PBMCs) was also repressed by AS-703026. At the molecular level, we showed that AS-703026 blocked LPS-induced MEK/ERK activation in above macrophages/monocytes. However, restoring ERK activation in AS-703026-treated RAW 264.7 cells by introducing a constitutive-actively (CA)-ERK1 only partially reinstated LPS-mediated TNFα production. Meanwhile, AS-703026 could still inhibit TNFα response in ERK1/2-depleted (by shRNA) RAW 264.7 cells. Significantly, we found that AS-703026 inhibited LPS-induced nuclear factor κB (NFκB) activation in above macrophages and COPD patients’ PBMCs. In vivo, oral administration of AS-703026 inhibited LPS-induced TNFα production and endotoxin shock in BALB/c mice. Together, we show that AS-703026 in vitro inhibits LPS-induced TNFα production in macrophages/monocytes, and in vivo protects mice from LPS-induced endotoxin shock. Thus, it could be further studied as a useful anti-inflammatory therapy for COPD patients.     

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.     

Ornithine-containing lipids stimulate CD14-dependent TNF-alpha production from murine macrophage-like J774.1 and RAW 264.7 cells

The ornithine-containing lipids (OL)-induced cytokine production pattern in macrophage-like J774.1 and RAW 264.7 cells was different from that in the peritoneal macrophages previously reported. OLs, as well as lipopolysaccharide (LPS) of Escherichia coli, strongly induced tumor necrosis factor (TNF) alpha but not interleukin (IL)-1beta in J774.1 cells. In the RAW cells, IL-1beta, TNF-alpha and prostaglandin E(2) were strongly induced by the OLs and LPS. OL- and serine-glycine-containing lipid (SGL)-induced TNF-alpha production in J774.1 and RAW 264.7 cells required serum. However, in CD14-deficient LR-9 cells, TNF-alpha was not induced by the OLs in the presence or absence of serum. OLs and a SGL almost completely inhibited the binding of (125)I-LPS to J774.1 cells. These results suggested that OLs and SGL activate macrophages via the CD14-dependent pathway.     

Anti-inflammatory effect of pyroglutamyl-leucine on lipopolysaccharide-stimulated RAW 264.7 macrophages

Aims

Food-derived peptides have been reported to yield a variety of health promoting activities. Pyroglutamyl peptides are contained in the wheat gluten hydrolysate. In the present study, we investigated the effect of pyroglutamyl dipeptides on the lipopolysaccharide (LPS)-induced inflammation in macrophages.

Main methods

RAW 264.7 macrophages were treated with LPS and various concentrations of pyroglutamyl-leucine (pyroGlu-Leu), -valine (pyroGlu-Val), -methionine (pyroGlu-Met), and -phenylalanine (pyroGlu-Phe). Cell viability/proliferation and various inflammatory parameters were measured by the established methods including ELISA and western blotting. The binding of fluorescein isothiocyanate-labeled LPS to RAW 264.7 cells was also measured fluorescently.

Key findings

All the tested dipeptides significantly inhibited the secretion of nitric oxide, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 from LPS-stimulated RAW 264.7 macrophages. Above all, pyroGlu-Leu inhibited the secretion of all these inflammatory mediators even at the lowest dose (200 μg/ml). PyroGlu-Leu dose-dependently suppressed IκBα degradation and MAPK (JNK, ERK, and p38) phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, it did not affect the binding of LPS to the cell surface.

Significance

Our results indicated that pyroGlu-Leu inhibits LPS-induced inflammatory response via the blocking of NF-κB and MAPK pathways in RAW 264.7 macrophages.     

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.     

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.     

Mechanisms of antimicrobial and antiendotoxin activities of a triazine-based amphipathic polymer

TZP4 is a triazine-based amphipathic polymer designed to mimic the amphipathic structure found in antimicrobial peptides. TZP4 showed potent antimicrobial activity comparable to melittin against antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa. TZP4 showed high resistance to proteolytic degradation and low tendency to develop drug resistance. The results from membrane depolarization, SYTOX Green uptake, flow cytometry, and gel retardation revealed that the mechanism of antimicrobial action of TZP4 involved an intracellular target rather than the bacterial cell membrane. Furthermore, TZP4 suppressed the messenger RNA levels of inducible nitric oxide synthase and tumor necrosis factor-α (TNF-α) and inhibited the release of nitric oxide and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. BODIPY-TR-cadaverine displacement and dissociation of fluorescein isothiocyanate (FITC)-labeled LPS assays revealed that TZP4 strongly bound to LPS and disaggregated the LPS oligomers. Flow cytometric analysis demonstrated that TZP4 inhibits the binding of FITC-conjugated LPS to RAW264.7 cells. These observations indicate that TZP4 may exert its antiendotoxic activity by directly binding with LPS and inhibiting the interaction between LPS and CD14⁺ cells. Collectively, TZP4 is a promising drug candidate for the treatment of endotoxic shock and sepsis caused by Gram-negative bacterial infections.     

The nuclear IkappaB protein IkappaBNS selectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of the colonic lamina propria

Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPMphi). CLPMphi of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine production and costimulatory molecule expression. We compared CLPMphi gene expression profiles of wild-type mice with IL-10-deficient mice, and identified genes that are selectively expressed in wild-type CLPMphi. These genes included nuclear IkappaB proteins such as Bcl-3 and IkappaBNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-alpha production, we analyzed the role of IkappaBNS in macrophages. Lentiviral introduction of IkappaBNS resulted in impaired LPS-induced IL-6 production, but not TNF-alpha production in the murine macrophage cell line RAW264.7. IkappaBNS expression led to constitutive and intense DNA binding of NF-kappaB p50/p50 homodimers. IkappaBNS was recruited to the IL-6 promoter, but not to the TNF-alpha promoter, together with p50. Furthermore, small interference RNA-mediated reduction in IkappaBNS expression in RAW264.7 cells resulted in increased LPS-induced production of IL-6, but not TNF-alpha. Thus, IkappaBNS selectively suppresses LPS-induced IL-6 production in macrophages. This study established that nuclear IkappaB proteins differentially regulate LPS-induced inflammatory cytokine production in macrophages.     

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.     

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.     

Antimicrobial peptide P18 inhibits inflammatory responses by LPS- but not by IFN-γ-stimulated macrophages

Antimicrobial peptide P18 markedly inhibited the expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells, whereas magainin 2 did not inhibit these activities. P18 dose-dependently reduced nitric oxide (NO) production by LPS-stimulated RAW 264.7 macrophage cells, with complete inhibition at 20 microg P18 ml(-1). In contrast, P18 had no effect on NO production and the expression of iNOS mRNA and iNOS protein by interferon-gamma (IFN-gamma)-stimulated RAW264.7 cells, suggesting P18 selectively inhibits LPS-stimulated inflammatory responses in macrophages. An LAL assay showed that P18 has strong LPS-neutralizing activity, indicating that P18 inhibits the inflammatory responses in LPS-stimulated macrophages by direct binding to LPS. Collectively, our results indicate that P18 has promising therapeutic potential as a novel anti-inflammatory as well as antimicrobial agent.     

Lipopolysaccharide-induced monocyte chemotactic protein-1 is enhanced by suppression of nitric oxide production, which depends on poor CD14 expression on the surface of skeletal muscle

It is known that lipopolysaccharide (LPS)-induced monocyte chemotactic protein (MCP)-1 secretion from tissues recruits monocytes from the circulation, but the mechanism of the LPS-induced MCP-1 production in skeletal muscle is largely unexplained. To clarify the effect of LPS on MCP-1 production in skeletal muscle cells, C2C12 cells from a mouse skeletal muscle cell line, and RAW 264.7 cells from a mouse macrophage cell line, were used to assess production of LPS-induced MCP-1, nitric oxide (NO) and interferon (IFN)-beta. In addition, we evaluated inducible NO synthases (iNOS) mRNA expression using RT-PCR, and cell surface expression of CD14 and toll-like receptor (TLR) 4 using flow cytometry. In C2C12 cells, LPS stimulation increased MCP-1 production (p < 0.01), but combined treatment with LPS and NO inducer, diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate (NONOate), significantly inhibited its production (p < 0.01). LPS stimulation neither induced production of NO nor of IFN-beta, which is an NO inducer. Recombinant IFN-beta stimulation, on the other hand, enhanced LPS-induced NO production (p < 0.01). Interestingly, we found that surface expression of CD14, which regulates IFN-beta production, in C2C12 cells was much lower than that in RAW 264.7 cells, although TLR4 expression on C2C12 cells was similar to that on RAW 264.7 cells. These data suggest that the reduced NO production in response to LPS may depend on low expression of CD14 on the cell surface of skeletal muscle, and that it may enhance LPS-induced MCP-1 production. Together, these functions of skeletal muscle could decrease the risk of bacterial infection by recruitment of monocytes.