Nitric oxide production by murine spleen cells stimulated with lipopolysaccharide from Actinobacillus actinomycetemcomitans

The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could induce murine spleen cells to produce nitric oxide (NO). Spleen cells derived from Balb/c mice were stimulated with LPS-A. actinomycetemcomitans or LPS from Escherichia coli for 4 days. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B, and cytokines (IFN-gamma and IL-4) on the production of NO were also assessed. The NO production from the carrageenan-treated spleen cells stimulated with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma was determined. The carrageenan-treated mice were transferred with splenic macrophages and the NO production was assessed from the spleen cells stimulated with LPS-A. actinomycetemcomitans or LPS-A. actinomycetemcomitans and IFN-gamma. The results showed that NO production was detectable in the cultures of spleen cells stimulated with LPS-A. actinomycetemcomitans in a dose-dependent fashion, but was lower than in the cells stimulated with LPS from E. coli. The NO production was blocked by NMMA and polymyxin B. IFN-gamma up-regulated but IL-4 suppressed the production of NO by the spleen cells stimulated with LPS-A. actinomycetemcomitans. The carrageenan-treated spleen cells failed to produce NO after stimulation with LPS-A. actinomycetemcomitans or both LPS-A. actinomycetemcomitans and IFN-gamma. Adoptive transfer of splenic macrophages to the carrageenan-treated mice could restore the ability of the spleen cells to produce NO. The results of the present study suggest that LPS-A. actinomycetemcomitans under the regulatory control of cytokines induces murine spleen cells to produce NO and that splenic macrophages are the cellular source of the NO production. Therefore, these results may support the view that NO production by LPS-A. actinomycetemcomitans-stimulated macrophages may play a role in the course of periodontal diseases.     

Anti‐inflammatory and anti‐endotoxin properties of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti‐inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)‐induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti‐inflammatory and anti‐endotoxin activities of Os and Os‐C, peptides derived from the carboxy‐terminal of a tick defensin, were investigated. Both Os and Os‐C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin‐binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os‐C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os‐C showed no scavenging activity. Os and Os‐C inhibited LPS/IFN‐γ induced NO and TNF‐α production in RAW 264.7 cells in a concentration‐dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF‐α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os‐C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os‐C, both peptides have in addition anti‐inflammatory and anti‐endotoxin properties. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

J774 macrophage-like cell line cytokine and chemokine patterns are modulated by <Emphasis Type="Italic">Francisella tularensis</Emphasis> LVS strain infection

Mutual interactions were investigated between intracellular parasitic bacterium Francisella tularensis (F.t.; highly virulent bacterium responsible for tularemia, replicating within the host macrophages) and murine macrophage-like cell line J774. Recombinant murine lymphokine INF-γ and/or LPS derived from E. coli were determined to stimulate in vitro antimicrobial activity of macrophage-like J774 cell line against the live vaccine strain (LVS) of F.t. through their ability to produce proinflammatory cytokines and chemokines. F.t. infection up-regulated IL-12 p40 production and down-regulated TNF-α production by stimulated macrophages; on the other hand, F.t. infection did not affect the production of IL-8, IL-6, MCP-5, and RANTES by stimulated macrophages. This showed that F.t. infection modulates the cytokine synthesis by J774 macrophage cell line.     

Phytochemical Profiling and Compound Isolation of Cissus rhombifolia Vahl. Leaves Aqueous Methanolic Extract with the Evaluation of Its Anti-Inflammatory Effect Using Lipopolysaccharide-Induced Inflammation in RAW 264.7 Cells

The inflammatory disorders represent a serious health issue. Certain Cissus species possess anti-inflammatory effect. Cissus rhombifolia Vahl. leaves’ anti-inflammatory activities and phytoconstituents are poorly characterized. In this study, 38 constituents were tentatively characterized in Cissus rhombifolia Vahl. leaves’ aqueous methanolic extract (CRLE) using high-performance liquid chromatography combined with mass spectrometry (HPLC/MS) and Proton Nuclear Magnetic Resonance (¹H-NMR). Myricetin, β-amyrin, and alliospiroside A, were isolated from CRLE using column chromatography. The anti-inflammatory effect of CRLE and its isolated compounds were studied in lipopolysaccharide (LPS)-induced RAW 264.7 cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) was used to assess how CRLE and its isolated compounds affected cell viability. Further, its effects on the production of intracellular NO, and inflammatory cytokines cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) were assessed by the Griess test, and cytokine enzyme-linked immunosorbent assays, respectively. CRLE and its isolated compounds, myricetin, β-amyrin, and alliospiroside A decreased the NO production. Western blotting was performed to assess the protein expression levels of the inflammatory cytokines inducible nitric oxide synthase (iNOS). Alliospiroside A downregulated IL-6, TNF-α, and COX-2 and inhibited the expression of iNOS. CRLE and its compounds represent effective alternative candidate to treat inflammatory diseases.     

In vitro effects of cAMP-elevating agents and glucocorticoid either alone or in combination on the production of nitric oxide,interleukin-12 and interleukin-10 in IFN-gamma- and LPS-activated mouse peritoneal macrophages

The effects of cAMP-elevating agents,N ⁶-2′-O-dibutyryl cAMP (Bu₂cAMP), and glucocorticoid (dexamethasone) on the production of inflammatory mediators—nitric oxide and interleukin-12 (IL-12) and anti-inflammatory mediator interleukin-10 (IL-10) were demonstrated in murine peritoneal macrophages. Inducible nitric oxide synthase (iNOS) and iNOS mRNA were detected by northern blot and western blot, respectively. The cAMP elevating agents Bu₂cAMP and prostaglandin E₂ each alone did not show any effect on NO production but along with IFN-γ and lipolysaccharide (LPS) they slightly enhanced NO production. Dexamethasone inhibited NO production in IFN-γ-and LPS-treated cells; cAMP elevating agents interfered with the NO production inhibited by dexamethasone. Inhibition was revealed at the mRNA level as well as at protein level. Bu₂cAMP or dexamethasone either alone or synergistically inhibited IL-12 production; Bu₂cAMP interfered with dexamethasone-mediated inhibition of IL-10 production in IFN-γ-and LPS-treated macrophages. The use of glucocorticoids along with cAMP elevating agents was beneficial in lowering the level of inflammatory mediator IL-12 and producing high levels of the anti-inflammatory mediator IL-10 active in cell protection. On the other hand, inteference of Bu₂cAMP with dexamethasone-mediated NO inhibition may have adverse effect. Therefore, adverse effects due to cAMP-mediated interference (inhibition) with NO synthesis may occur in many inflammatory diseases during combined drug therapy by glucocorticoids and cAMP elevating agents.     

Nitric oxide production by a murine macrophage cell line (RAW264.7 cells) stimulated with Aggregatibacter actinomycetemcomitans surface-associated material

Nitric oxide (NO) may play a crucial role in the pathogenesis of periodontal disease and, hence, the aim of the present study was to test the hypothesis that Aggregatibacter actinomycetemcomitans surface-associated material (SAM) stimulates inducible nitric oxide synthase (iNOS) activity and NO production by the murine macrophage cell line RAW264.7. Cells were stimulated with untreated or heat-treated A. actinomycetemcomitans SAM and with or without pre-treatment with l-N⁶-(1-Iminoethyl)-lysine (L-NIL) (an iNOS inhibitor), polymyxin B, interferon-gamma (IFN-γ) and Interleukin-4 (IL-4), IL-10, genistein [a protein tyrosine kinase (PTK) inhibitor], bisindolylmaleimide [a protein kinase C (PKC) inhibitor], bromophenacyl bromide (BPB) [a phospholipase A₂ (PLA2) inhibitor] or wortmannin [phosphatidylinositol 3-kinase (PI-3K) inhibitor]. The iNOS activity and nitrite production in the cell cultures were determined. Untreated but not heat-treated A. actinomycetemcomitans SAM-stimulated both iNOS activity and nitrite production in RAW264.7 cells. l-NIL, IL-4, IL-10, genistein, bisindolylmaleimide, or BPB, suppressed but IFN-γ enhanced both iNOS activity and nitrite production by A. actinomycetemcomitans SAM-stimulated cells. Wortmannin and polymyxin B failed to alter both iNOS activity or nitrite production by A. actinomycetemcomitans SAM treated cells. Therefore, the present study suggests that a heat-sensitive protein constituent(s) of A. actinomycetemcomitans SAM stimulates both iNOS activity and nitrite production by RAW264.7 cells in a cytokine, PTK, PKC, and PLA₂ but not PI-3K-dependent fashion.     

Endotoxin and staphylococcus aureus enterotoxin a induce different patterns of cytokines

The effects of Staphylococcus aureus enterotoxin A (SEA) and lipopolysaccharide (LPS) in cytokine production were assessed at the single cell level in cells obtained from healthy blood donors. Cytokine production was studied with UV-microscopy of fixed and permeabilized cells stained with cytokine specific monoclonal antibodies. The cytokines evaluated included tumour necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-10, IL-2, IL-4, interferon (IFN)-γ and TNF-β. LPS exhibited marked production of IL-1α, IL-1β, TNF-α, IL-6 and IL-8. After LPS stimulation IL-1α, IL-1β, TNF-α and IL-8 were the dominating products, all peaking at or before 4 hours after cell stimulation. In addition, IL-10 production was evident after 12 hours of cell stimulation. The T-lymphocyte-derived cytokines TNF-β, IL-2, IFN-γ and IL-4 were never detected in the cultures. All cytokine production, except IL-8, was downregulated at 96 hours.In contrast, peak production of IL-1α, IL-1β and IL-8, which were the dominant products, occurred after 12 hours in the SEA-stimulated cultures. Further, a significant T-lymphocyte production of TNF-β, TNF-α, IFN-γ and IL-2 was found with peak production 12–48 hours after initiation. Only low amounts of IL-6 were evident.The two types of cytokine pattern and kinetics found may correspond to the different clinical conditions after invasive Gram-negative Escherichia coli vs Gram-positive Staphylococcus aureus infections in humans, with a much more rapid onset of disease after E. coli infections. The data may also imply that different immunomodulating approaches should be considered in life-threatening diseases with the two microbacterial agents.     

Immunostimulatory Effect of Enzyme-Modified <Emphasis Type="Italic">Hizikia fusiforme</Emphasis>in a Mouse Model In Vitro and Ex Vivo

Hizikia fusiforme, a brown seaweed, has been utilized as a health food and in traditional medicine. In this study, we investigated whether enzyme-modified H. fusiforme extracts (EH) have immunological effects compared with normal H. fusiforme extracts (NH). The effects of NH and EH on immune responses were investigated by assessing nitric oxide (NO) production, phagocytosis, and cytokine secretion in RAW 264.7 murine macrophages and mice. Also, fucosterol was evaluated to find the active component of NH and EH by addressing cytotoxicity test and NO production. Both of NH and EH significantly increased cell viability and NO synthesis. Tumor necrosis factor-α (TNF-α) expression was more induced by EH with LPS treatment. Phagocytic activity, as the primary function of macrophages, was markedly induced by EH treatment. Additionally, EH encouraged splenocyte proliferation and recovered the levels of cytokines IL-1β, IL-6, and TNF-α in mice. Finally, fucosterol increased NO production with no cytotoxicity, which means that fucosterol is an active component of EH. In conclusion, EH has the potential to modulate immune function and could offer positive therapeutic effect for immune system diseases.     

Dynamics of antagonistic potency of <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> PG lipopolysaccharide against endotoxin-induced effects

The dynamics of antagonistic potency of lipopolysaccharide (LPS) isolated from Rhodobacter capsulatus PG on the synthesis of proinflammatory (TNF-α, IL-1β, IL-8, IL-6, IFN-γ) and antiinflammatory (IL-10, IL-1Ra) cytokines induced by highly stimulatory endotoxins from Escherichia coli or Salmonella enterica have been studied. Using human whole blood, we have shown that R. capsulatus PG LPS inhibited most pronouncedly the endotoxin-induced synthesis of TNF-α, IL-1β, IL-8, and IL-6 during the first 6 h after endotoxin challenge. Similarly, the endotoxin-induced release of IFN-γ was abolished by R. capsulatus PG LPS as well (24 h). In contrast to the above-mentioned cytokines, the relatively weak antagonistic activity of R. capsulatus PG LPS against endotoxin-triggered production of IL-6 and IL-8 was revealed. Since R. capsulatus PG LPS displays more potent antagonistic activity against deleterious effects of S. enterica LPS than those of E. coli LPS in the cases of such cytokines as IL-1β (6 and 24 h), IL-6 and IL-8 (4 h), we conclude that the effectiveness of protective action of antagonist is mostly determined by the primary lipid A structure of the employed agonist.     

TNF-α、IL-1β及LPS对血管平滑肌细胞一氧化氮合酶基因表达的影响及作用机制研究

通过ＲＮＡ印迹分析和亚硝酸盐含量测定检查ＴＮＦ－α、ＩＬ－１β和ＬＰＳ对大鼠血管平滑肌细胞（ＶＳＭＣ）诱导型一氧化氮合酶（ｉＮＯＳ）基因表达及ＮＯ生成的影响．结果表明,ＴＮＦ－α、ＩＬ－１β和ＬＰＳ均能显著诱导ＶＳＭＣｉＮＯＳ基因表达和促进ＮＯ生成,其作用强度与浓度和作用时间有关;双因素（ＴＮＦ－α＋ＬＰＳ,ＬＰＳ＋ＩＬ－１β）对诱导ｉＮＯＳ基因表达及ＮＯ生成产生协同作用．ＰｏｌｙｍｙｘｉｎＢ和地塞米松可部分抑制ＴＮＦ－α对ｉＮＯＳ基因表达的诱导作用及ＮＯ生成     

Design, synthesis, and evaluation of a new fluorescent probe for measuring polymyxin-lipopolysaccharide binding interactions

Fluorescence assays employing semisynthetic or commercial dansyl-polymyxin B have been widely employed to assess the affinity of polycations, including polymyxins, for bacterial cells and lipopolysaccharide (LPS). The five primary γ-amines on diaminobutyric acid residues of polymyxin B are potentially derivatized with dansyl-chloride. Mass spectrometric analysis of the commercial product revealed a complex mixture of di- or tetra-dansyl-substituted polymyxin B. We synthesized a mono-substituted fluorescent derivative, dansyl[Lys]¹polymyxin B₃. The affinity of polymyxin for purified gram-negative LPS and whole bacterial cells was investigated. The affinity of dansyl[Lys]¹polymyxin B₃ for LPS was comparable to polymyxin B and colistin, and considerably greater (K_d < 1 μM) than for whole cells (K_d ∼ 6–12 μM). Isothermal titration calorimetric studies demonstrated exothermic enthalpically driven binding between both polymyxin B and dansyl[Lys]¹polymyxin B₃ to LPS, attributed to electrostatic interactions. The hydrophobic dansyl moiety imparted a greater entropic contribution to the dansyl[Lys]¹polymyxin B₃–LPS reaction. Molecular modeling revealed a loss of electrostatic contact within the dansyl[Lys]¹polymyxin B₃–LPS complex due to steric hindrance from the dansyl[Lys]¹ fluorophore; this corresponded with diminished antibacterial activity (MIC ?16 μg/mL). Dansyl[Lys]¹polymyxin B₃ may prove useful as a screening tool for drug development.     

Pulsatilla decoction and its active ingredients inhibit secretion of NO,ET‐1, TNF‐α, and IL‐1α in LPS‐induced rat intestinal microvascular endothelial cells

To investigate the pharmacological mechanism of the traditional Chinese medicine, Pulsatilla decoction (PD), the levels of nitric oxide (NO), endothelin‐1 (ET‐1), tumor necrosis factor‐α (TNF‐α), and interleukin‐1α (IL‐1α) secreted by cultured rat intestinal microvascular endothelial cells (RIMECs) were determined after treatment with PD and its seven active ingredients, namely anemoside B₄, anemonin, berberine, jatrorrhizine, palmatine, aesculin, and esculetin. RIMECs were challenged with lipopolysaccharide (LPS) at 1 µg ml^?1 for 3 h and then treated with PD at 1, 5, and 10 mg ml^?1 and its seven ingredients at 1, 5, and 10 µg ml^?1 for 21 h, respectively. The results revealed that PD, anemonin, berberine, and esculetin inhibited the production of NO; PD, anemonin, and esculetin inhibited the secretion of ET‐1; PD, anemoside B₄, berberine, jatrorrhizine, and aesculin downregulated TNF‐α expression; PD, anemoside B₄, berberine, and palmatine decreased the content of IL‐1α. It showed that PD and its active ingredients could significantly inhibit the secretion of NO, ET‐1, TNF‐α, and IL‐1α in LPS‐induced RIMECs and suggested they would reduce inflammatory response via these cytokines. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of Female Hormones (17β-Estradiol and Progesterone) on Nitric Oxide Production by Alveolar Macrophages in Rats

The effect of female sex hormones on nitric oxide (NO) production was studied in alveolar macrophages (AMs). Male rats were treated with endotoxin (LPS) intratracheally or saline as control. AMs were obtained by bronchoalveolar lavage 90 min later and were cultured in the presence or in the absence of LPS and 17β-estradiol or progesterone (10⁻⁹to 10⁻⁴M). NO production was assessed by measurement of nitrites in the medium. In some experiments, NO production by AMs was measured in intratracheally LPS-treated orchidectomized rats or in female control and ovariectomized rats. Both spontaneous and stimulated NO production were higher in AMs from female than from male rats, but without statistical significance. However, ovariectomy induced significant inhibition in spontaneous production of NO by AMs. In orchidectomized rats, the NO response by AMs to LPS stimulation relative to spontaneous NO production was significantly downregulated. Female sex hormones in physiological concentrations seem to be necessary for spontaneous NO production in female rats. Pharmacological doses of estradiol inhibitedin vitroLPS-stimulated NO production in AMs of both saline- and LPS-treated rats, and basal NO production only in LPS-treated male rats. Progesterone at 10⁻⁴M inhibited basal andin vitroLPS-stimulated NO generation by AMs of both saline- and LPS-treated male rats. In LPS-treated female ratsin vitroLPS-stimulated NO production was not affected by estradiol treatment. In ovariectomized LPS-treated female rats progesterone at 10⁻⁵M significantly inhibited NO production byin vitro-stimulated AMs. Thus female sex hormones may contribute to the gender-related differences in the immune response.     

The Effect of Lipopolysaccharide from <Emphasis Type="Italic">Proteus mirabilis</Emphasis> on the Level of the Stable End Metabolic Products of Nitric Oxide in Blood Platelets

Nitric oxide (^•NO) plays an important role in a number of physiologic processes. Evidence exists that ^•NO, which stimulates soluble guanylate cyclase and enhances cyclic guanosine monophosphate (cGMP) levels, may inhibit platelet activation. In contrast, during platelet activation induced by different agonists, synthesis of ^•NO in platelets occurs. In these studies, production of the stable end-products of ^•NO-nitrite and nitrate (NO_x) in human platelets, stimulated by different doses of lipopolysaccharide from Proteus mirabilis (LPS; endotoxin), has been evaluated. LPS is a weak platelet agonist that may activate various steps of platelet activation with the generation of reactive oxygen species. The mechanism of platelet activation induced by the endotoxin is not known. The aim of the present study was to measure the level of nitrite and NO_x in blood platelets treated with LPS and to examine the level of nitrotyrosine in platelet proteins caused by LPS. Our results show that LPS at a low concentration (6.8 ng/ml) caused a decrease (approximately 80%) in the NO_x level, whereas at higher concentrations (13.6 and 25 ng/ml) it induced an increase in the NO_x level (approximately 210% and 260%, respectively). Our results indicate that LPS, like other agonists (thrombin, platelet-activating factor), can stimulate ^•NO production in platelets. After incubating platelets with LPS, we also observed a distinct increase in platelet protein nitration (3-nitrotyrosine).     

Effects of pro-inflammatory cytokines,lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE₂, PGF_2α) or their corresponding ethanolamides (PGE₂-EA or PGF_2α-EA) over 48 h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20 h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10⁻⁵ M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10⁻⁴ M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE₂ transiently increased Caco-2 monolayer permeability at 24 h, while LPS (10 μg/ml) increased permeability over 24–48 h.These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE₂ transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.     

ApoE Production in Human Monocytes and Its Regulation by Inflammatory Cytokines

The apoE production by tissue macrophages is crucial for the prevention of atherosclerosis and the aim of this study was to further elucidate how this apolipoprotein is regulated by cytokines present during inflammation. Here we studied apoE production in peripheral blood mononuclear cells (PBMC) and analysis was made with a newly developed apoE ELISpot assay. In PBMC, apoE secretion was restricted to monocytes with classical (CD14⁺⁺CD16⁻) and intermediate (CD14⁺CD16⁺) monocytes being the main producers. As earlier described for macrophages, production was strongly upregulated by TGF-β and downregulated by bacterial lipopolysaccharide (LPS) and the inflammatory cytokines IFN-γ, TNF-α and IL-1β. We could here show that a similar down-regulatory effect was also observed with the type I interferon, IFN-α, while IL-6, often regarded as one of the more prominent inflammatory cytokines, did not affect TGF-β-induced apoE production. The TNF-α inhibitor Enbrel could partly block the down-regulatory effect of IFN-γ, IFN-α and IL-1β, indicating that inhibition of apoE by these cytokines may be dependent on or synergize with TNF-α. Other cytokines tested, IL-2, IL-4, IL-12, IL-13, IL-17A and IL-23, had no inhibitory effect on apoE production. In contrast to the effect on monocytes, apoE production by primary hepatocytes and the hepatoma cell line HepG2 was more or less unaffected by treatment with cytokines or LPS.     

Normal rat hepatic stellate cells respond to endotoxin in LBP-independent manner to produce inhibitor(s) of DNA synthesis in hepatocytes

Endotoxin is implicated in the pathology of acute liver failure. The mechanisms of its actions on quiescent hepatic stellate cells (qHSCs) and their implications in hepatocyte injury are incompletely understood. We investigated effects of endotoxin (bacterial lipopolysaccharide; LPS) on qHSCs and subsequently on hepatocytes. After overnight culture following their isolation, qHSCs were incubated with or without endotoxin for 24 h. The cells and the culture supernatant were analyzed for cytokines and nitric oxide (NO) synthesis. The effects of qHSC-conditioned media on hepatocytes were then determined. LPS increased inducible NO synthase expression, stimulated NO synthesis, and inhibited DNA synthesis in qHSCs. qHSC-conditioned medium inhibited DNA synthesis in hepatocytes without affecting NO synthesis, while LPS (1-1,000 ng/ml)-conditioned qHSC medium stimulated NO synthesis and caused further inhibition of DNA synthesis and apoptosis. These effects of LPS were more pronounced when qHSCs were incubated with serum, but not with LPS-binding protein (LBP) although CD14 (a receptor for LPS-LBP complex) was found in qHSCs. LPS stimulated the synthesis of TNF-alpha, interleukin (IL)-6, and IL-1beta but not of TGF-beta in qHSCs. Individually or together, L-N(G)-monomethylarginine and antibodies to IL-1beta, IL-6, and TNF-alpha only partly reversed qHSC + LPS-conditioned medium-induced inhibition of DNA synthesis in hepatocytes. These results suggest that the effects of LPS on qHSCs are novel, occurring without the aid of LBP/CD14. They also indicate that other factors, in addition to NO, TGF-beta, TNF-alpha, IL-1beta, and IL-6 are involved in the mechanisms of the growth inhibitory effects of qHSCs on hepatocytes.     

Bupleurum Polysaccharides Attenuates Lipopolysaccharide-Induced Inflammation via Modulating Toll-Like Receptor 4 Signaling

Background

Bupleurum polysaccharides (BPs), isolated from Bupleurum smithii var. parvifolium, possesses immunomodulatory activity, particularly on inflammation. Bacterial endotoxin lipopolysaccharide (LPS) triggers innate immune responses through Toll-like receptor 4 (TLR4) on host cell membrane. The present study was performed to evaluate whether the therapeutic efficacy of BPs on suppression of LPS’s pathogenecity could be associated with the modulating of TLR4 signaling pathway.

Methodology/Principal Findings

LPS stimulated expression and activation of factors in the TLR4 signaling system, including TLR4, CD14, IRAK4, TRAF6, NF-κB, and JNK, determined using immunocytochemical and/or Western blot assays. BPs significantly inhibited these effects of LPS. LPS increased pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-12p40, and IFN-β) and NO production, evaluated using ELISA and Griess reaction assays, respectively. BPs antagonized these effects of LPS. Interestingly, BPs alone augmented secretion of some pro-inflammatory cytokines of non-LPS stimulated macrophages and enhanced phagocytic activity towards fluorescent E.coli bioparticles. In a rat model of acute lung injury (ALI) with pulmonary hemorrhage and inflammation, BPs ameliorated lung injuries and suppressed TLR4 expression.

Significance

The therapeutic properties of BPs in alleviating inflammatory diseases could be attributed to its inhibitory effect on LPS-mediated TLR4 signaling.     

Dynamics of cytokine production in human peripheral blood mononuclear cells stimulated by LPS or infected by Borrelia

To specify the role of individual cytokines in the immune response to pyrogens, isolated and cultivated human peripheral blood mononuclear cells (PBMC) were used for the experiments. Different pyrogens (lipopolysaccharide from Escherichia coli - LPS and live Borrelia afzelii) were applied and the time course of changes in concentrations of different cytokines in the medium was followed using the ELISA method. It was found that nonstimulated human PBMC proliferate under in vitro conditions and produce IL-6, TNF-alpha, IL-10 and finally also IL-1beta. Productions of IL-12 and INF-gamma are not changed. Proliferation of PBMC is potentiated after incubation with LPS or live Borrelia. PBMC stimulated by LPS increase the net production (stimulated minus unstimulated) of IL-1beta and TNF-alpha significantly, while production of IL-6 was smaller. A delayed increase in the production of IL-10 was also observed. Productions of IL-12 and INF-gamma were not influenced. In contrast to LPS, stimulation of PBMC with live Borrelia, increases also the production of IL-12 and IFN-gamma, besides IL-1beta, TNF-alpha, IL-6 and IL-10. Productions of IL-1beta, IL-6 and TNFalpha increased immediately after incubation with both LPS and Borrelia, while productions of IL-12 and INF-gamma begin to increase 8 hours and production of IL-10 12 hours after stimulation. Data indicate that stimulation with different pyrogens may activate the cells of the immune cascade in a different way. Stimulation of BPMC by LPS seems to activate the initial steps of the immune response (macrophages and granulocytes) only, while infection with live Borrelia also stimulates the later phase of the immune response, probably due to effect of initially produced cytokines.