5,6-Dihydroxyindole-2-carboxylic acid,a diffusible melanin precursor,is a potent stimulator of lipopolysaccharide-induced production of nitric oxide by J774 macrophages

Pre-incubation of J774 murine macrophages with 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a diffusible intermediate in the biosynthesis of eumelanins, leads to a marked increase in the levels of nitric oxide (NO) produced by lipopolysaccharide (LPS)-induced NO-synthase (iNOS). The effect varies with DHICA concentratior being maximum at a concentration of 1 × 10⁻⁶M, and is suppressed by the NOS inhibitor N^G-monomethyl-L-arginine (L-NMMA). No stimulation is observed when macrophages are exposed to DHICA after activation with LPS, indicating that the indole does not affect the catalytic activity of iNOS. These results point to a hitherto unrecognized role of DHICA as a chemical messenger mediating interaction between active melanocytes and macrophages in epidermal inflammatory and immune responses.     

Interleukin-10 (IL-10) inhibits the induction of nitric oxide synthase by interferon-gamma in murine macrophages.

A murine macrophage cell line, J774, expresses high levels of the enzyme nitric oxide synthase (NOS) and produces large amounts of nitric oxide (NO) when activated with recombinant interferon (IFN)-gamma and a low concentration of LPS (10 ng/ml). Both the expression of NOS and the production of NO were inhibited by recombinant IL-10 in a dose-dependent manner. The inhibition was effective only when the cells were pretreated with IL-10; addition of IL-10 at the same time or after IFN-gamma activation was without effect. These results demonstrate that IL-10, a product of Th2 (helper T lymphocyte 2) cells, can antagonise the function of IFN-gamma, a product of Th1 cells, by modulating the mechanism of synthesis of nitric oxide in the macrophages.     

Restoration of nitric oxide production by aldose reductase inhibitor in human endothelial cells cultured in high-glucose medium

The effects of elevated glucose and aldose reductase inhibitor (ARI:ONO-2235) on nitric oxide (NO) production in cultured human umbilical endothelial cells (HUVEC) were evaluated. Aldose reductase and nitric oxide synthase(NOS) share NADPH as an obligate cofactor, therefore it is suggested that the enhanced of glucose flux (27.5 mM) by aldose reductase inhibited NO production by blunting NOS activity. However, the addition of ONO-2235 (100 μM) prevented the inhibition of [NO_2⁻] production. Since ARI decreases glucose-mediated inhibition of NO production in HUVEC, this agent might ameliorate endothelial function associated with diabetes.     

Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism

Oxidative stress contributes to the pathogenesis of many disorders, including diabetes and cardiovascular disease. Immune cells are major sources of superoxide (O₂^∙−) as part of the innate host defense system, but exaggerated and sustained O₂^∙− generation may lead to progressive inflammation and organ injuries. Previous studies have proven organ-protective effects of inorganic nitrite, a precursor of nitric oxide (NO), in conditions manifested by oxidative stress and inflammation. However, the mechanisms are still not clear. This study aimed at investigating the potential role of nitrite in modulating NADPH oxidase (NOX) activity in immune cells. Mice peritoneal macrophages or human monocytes were activated by lipopolysaccharide (LPS), with or without coincubation with nitrite. O₂^∙− and peroxynitrite (ONOO⁻) formation were detected by lucigenin-based chemiluminescence and fluorescence techniques, respectively. The intracellular NO production was measured by DAF-FM DA fluorescence. NOX isoforms and inducible NO synthase (iNOS) expression were detected by qPCR. LPS increased both O₂^∙− and ONOO⁻ production in macrophages, which was significantly reduced by nitrite (10 µmol/L). Mechanistically, the effects of nitrite are (1) linked to increased NO generation, (2) similar to that observed with the NO donor DETA-NONOate, and (3) can be abolished by the NO scavenger carboxy-PTIO or by the xanthine oxidase (XO) inhibitor febuxostat. Nox2 expression was increased in activated macrophages, but was not influenced by nitrite. However, nitrite attenuated LPS-induced upregulation of iNOS expression. Similar to that observed in mice macrophages, nitrite also reduced O₂^∙− generation in LPS-activated human monocytes. In conclusion, XO-mediated reduction of nitrite attenuates NOX activity in activated macrophages, which may modulate the inflammatory response.     

Adaptation of macrophages to exercise training improves innate immunity

The effects of 3-week exercise training on the functions of peritoneal macrophages from BALB/c mice were investigated. Lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and proinflammatory cytokine production in macrophages from trained mice was markedly higher than those from control mice. Meanwhile, exercise training decreased the steady state level of β₂-adrenergic receptor (β₂AR) mRNA in macrophages. Overexpression of β₂AR in the macrophage cell line RAW264 by transfecting with β₂AR cDNA suppressed NO synthase (NOS) II expression but dose not influenced proinflammatory cytokine expression. When expression of transfected β₂AR in RAWar cells was downregulated by a tetracycline repressor-regulated mammalian expression system, NOS II mRNA expression was significantly increased; this suggested that the changes in the β₂AR expression level in macrophages associated with exercise training play a role in the regulation of NO production following LPS stimulation. These findings indicate that exercise training improves macrophage innate immune function in a β₂AR-dependent and -independent manner.     

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.     

Oleanolic acid analogs as NO,TNF-α and IL-1β inhibitors: Synthesis,biological evaluation and docking studies

A series of oleanolic acid analogs, characterized by structural modifications at position C-3 and C-28 of oleanane skeleton were synthesized and assessed for antiinflammatory potential towards lipopolysaccharide (LPS) induced nitric oxide (NO) production in macrophages. Results revealed that all the synthesized analogs of oleanolic acid inhibit NO production with an IC₅₀ of 2.66–41.7 μM as compared to the specific nitric oxide synthase (NOS) inhibitor, L-NAME (IC₅₀ = 69.21 and 73.18 μM on RAW 264.7 and J774A.1 cells, respectively) without affecting the cell viability when tested at their half maximal concentration. The most potent NO inhibitors (2, 8, 9 and 10) at a concentration of 20 μg/mL also demonstrated mild inhibition (27.9–51.9%) of LPS-induced tumor necrosis factor alpha (TNF-α) and weak inhibition (11.1–37.5%) towards interleukin 1-beta (IL-1β) production in both the cells. The present study paves a direction that analogs of oleanolic acid can be employed as a lead in the development of potent NO inhibitors. Molecular docking studies also showed that 10 (with top Goldscore docking pose 19.05) showed similar interaction as that of co-crystallized inhibitor and, thereby, helps to design the potent inhibitors of TNF-α.     

Prolonged lipopolysaccharide inhibits leukotriene synthesis in peritoneal macrophages: mediation by nitric oxide and prostaglandins

Resident rat peritoneal macrophages synthesize a variety of prostanoids and leukotrienes from arachidonic acid. Overnight treatment with lipopolysaccharide (LPS) induces the synthesis of cyclooxygenase-2 (COX-2) and an altered prostanoid profile that emphasizes the preferential conversion of arachidonic acid to prostacyclin and prostaglandin E2. In these studies, we report that exposure to LPS also caused a strong suppression of 5-lipoxygenase but not 12-lipoxygenase activity, indicated by the inhibition of synthesis of both leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE), but not of 12-HETE. Inhibition of 5-lipoxygenase activity by LPS was both time- and dose-dependent. Treatment of macrophages with prostaglandin E2 partially inhibited leukotriene synthesis, and cyclooxygenase inhibitors partially blocked the inhibition of leukotriene generation in LPS-treated cells. In addition to COX-2, nitric oxide synthase (NOS) was also induced by LPS. Treatment of macrophages with an NO donor mimicked the ability of LPS to significantly reduce leukotriene B4 synthesis. Inhibition of NOS activity in LPS-treated cells blunted the suppression of leukotriene synthesis. Inhibition of both inducible NOS and COX completely eliminated leukotriene suppression. Finally, macrophages exposed to prolonged LPS demonstrated impaired killing of Klebsiella pneumoniae and the combination of NOS and COX inhibitors restored killing to the control level. These results indicate that prolonged exposure to LPS severely inhibits leukotriene production via the combined action of COX and NOS products. The shift in mediator profile, to one that minimizes leukotrienes and emphasizes prostacyclin, prostaglandin E2 and NO, provides a signal that reduces leukocyte function, as indicated by impaired killing of Gram-negative bacteria.     

氧化修饰低密度脂蛋白对巨噬细胞一氧化氮合酶基因表达的影响

氧化修饰LDL(OX-LDL)可抑制脂多糖(LPS)诱导的巨噬细胞NO释放, 而正常(N-LDL)和乙酰化LDL(AC-LDL)则没有抑制作用.OX-LDL对NO释放的抑制作用随LDL修饰程度的升高而增强,且具有浓度和时间效应.狭缝杂交结果显示OX-LDL处理可使LPS诱导的巨噬细胞NOS mRNA含量下降,提示OX-LDL对NO释放的抑制作用可能发生在转录水平.     

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.     

Induction of nitric oxide synthase by saponins of heat-processed ginseng

Total saponin of heat-processed ginseng (TSHG) stimulated the production of nitric oxide (NO) in interferon-gamma (IFN-gamma)-primed macrophages through the increased expression of inducible nitric oxide synthase (iNOS). However, TSHG by itself had a very weak effect on the NO synthesis without IFN-gamma priming. The saponins of white ginseng inhibited the NO production in lipopolysaccharide (LPS)/IFN-gamma activated macrophages rather than the stimulation of NO production found in IFN-gamma primed macrophages. The NO production by TSHG-stimulated macrophages was inhibited by the NOS inhibitor (N(G)-monomethyl-L-arginine (L-NMMA)) and nuclear factor-kappaB inhibitor (pyrrolidine dithiocarbamate (PDTC)). TSHG showed different serum-dependence from LPS on the activation of IFN-gamma primed macrophages. This property of TSHG may explain the intensified anti-tumor properties of heat-processed ginseng through its immunostimulating activity.     

Regulation of prostaglandin E2 production by the superoxide radical and nitric oxide in mouse peritoneal macrophages

The purpose of this study was to elucidate the role of NO and O^-₂ on enzymatic components of cyclooxygenase (COX) pathway in peritoneal macrophages. Activation of murine peritoneal macrophages by lipopolysaccharides (LPS) resulted in time-dependent production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). This stimulation was also accompanied by the production of other reactive oxygen species such as superoxide (O^-₂), and by increased expression of COX-2. Our results provide evidence that O^-₂ may be involved in the pathways that result in arachidonate release and PGE₂ formation by COX-2 in murine peritoneal macrophages stimulated by LPS. However, we were not able to demonstrate that NO participates in the regulation of PG production under our experimental conditions.     

Lipopolysaccharide stimulates nitric oxide synthase-2 expression in murine skeletal muscle and C(2)C(12) myoblasts via Toll-like receptor-4 and c-Jun NH(2)-terminal kinase pathways

The inducible form of nitric oxide synthase (NOS2) catalyzes the synthesis of nitric oxide (NO) from arginine in response to injury and infection. NOS2 is expressed predominantly by macrophages and lymphocytes. However, skeletal muscle also expresses NOS2 in response to inflammatory stimuli. The present study sought to determine whether lipopolysaccharide (LPS) stimulates NOS2 in skeletal muscle via Toll-like receptor-4 (TLR4). Intraperitoneal injection of LPS in wild-type mice (C3H/HeSnJ) increased NOS2 mRNA fourfold in skeletal muscle, while no change in NOS2 mRNA was observed in C3H/HeJ mice that harbored a mutation in the LPS receptor. NOS2 coimmunoprecipitated with the muscle-specific caveolin-3 protein, suggesting that myofibers per se respond to LPS in vivo. LPS stimulated NOS2 mRNA expression in C₂C₁₂ myocytes, and the regulation of NOS2 mRNA was comparable in myoblasts and differentiated myotubes. LPS transiently stimulated the phosphorylation of the interleukin-1 receptor-associated kinase (IRAK-1) in C₂C₁₂ cells and decreased the total amount of IRAK-1 both in vitro and in vivo over time. LPS stimulated the expression of an NF- reporter plasmid, and this was inhibited by the proteasomal inhibitor MG-132. Both myoblasts and myotubes expressed TLR2 and TLR4 mRNA. Expression of a dominant negative form of TLR4 in C₂C₁₂ cells blocked LPS-induced NF- reporter activity. SP-600125 [a c-Jun NH₂-terminal kinase (JNK) inhibitor] also prevented LPS stimulation of NOS2 expression. Moreover, the JNK inhibitor prevented the LPS-induced increase in NO synthesis. These data indicate that LPS increases NOS2 mRNA expression in muscle via a TLR4-dependent mechanism. interleukin-1 receptor-associated kinase; myotube; interleukin; dominant negative     

Paramecium caudatum as a source of nitric oxide: Chemiluminescent detection based on Bluestar® Forensic reagent connected with microdialysis

Nitric oxide (NO) chemistry inside the body is the most interesting part of its behavior. NO is involved in controlling blood pressure, and in transmitting nerve signals and a variety of other signaling processes. To explain the behavior of NO, it is necessary to determine its immediate concentration or observe time‐dependent changes in its concentration. In Paramecium caudatum, NO is formed by calcium‐dependent nNOS (NOS1)‐like protein, which is distributed in the cytoplasm. NO synthesis affects the ciliary beat and consequent motility of cells and blocked NO synthesis reduces the ability of cells to move. The possibility of online coupling of microdialysis (of P. caudatum solution) with NO detection is demonstrated. Direct measurement of NO is carried out using dilute Bluestar^® Forensic reagent (luminol–H₂O₂ system; one of the NO detections is based upon the chemiluminescent reaction between NO and the luminol–H₂O₂ system, which is specifically reactive to NO). The effect of a nitric oxide synthase inhibitor, NG‐nitro‐l ‐arginine methyl ester was observed. NO production was inhibited and the movement of P. caudatum was restricted. These effects were time dependent and after a specific time were reversed.     

Quantitative nitric oxide production by rat, bovine and porcine macrophages

The aim of this work was to compare in vitro nitric oxide (NO) production by rat, bovine and porcine macrophages. NO production was induced by lipopolysaccharide (LPS) or by phorbol 12-myristate 13-acetate (PMA) with ionomycin or recombinant interferon gamma (rIFN-γ) and was assessed by Griess reaction. NO synthase type II (NOS II) expression was quantified by immunocytochemistry, Western blot and real-time polymerase chain reaction (RT-PCR). There were differences in NO production by pulmonary alveolar macrophages (PAM) in all species tested. The largest amounts of NO were produced by rat PAM. Less NO was produced by bovine PAM. Moreover, PAM in rats and cows differed in their abilities to respond to various stimulators. Neither porcine PAM nor Kupffer cells produced NO. Stimulation of porcine PAM with alternative concentrations of LPS did not lead to inducing NO production. Stimulation of porcine PAM with rIFN-γ together with LPS led to a significant increase in the expression of NOS II mRNA, albeit without detectable NO production or NOS II expression on the protein level.     

Inhibitory effects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line,RAW 264.7, activated by lipopolysaccharide and interferon-γ

An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC₅₀ of 10.7 μM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-γ for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[¹⁴C]arginine to L-[¹⁴C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity.     

Retinoic acid-induced differentiation in a human neuroblastoma cell line is associated with an increase in nitric oxide synthesis

The human neuroblastoma cell line SK-N-BE, after incubation with 10 μM retinoic acid (RA) or 20 nM phorbol 12-myristate 13-acetate (PMA), underwent biochemical and morphological signs of differentiation within 10–14 days. In parallel, SK-N-BE cells produced significantly higher amounts of nitric oxide (NO) in comparison with controls, as assessed by the measurement of nitrite and nitrate in the culture supernatant and of NO synthase (NOS) activity in the cell lysates (measured as ability to convert [³H]arginine into [³H]citrulline and as NADPH diaphorase activity). Nitrite/nitrate production was abolished by adding the NO scavenger hemoglobin in the culture medium and was inhibited by aminoguanidine (AG, a selective inhibitor of the inducible NOS isoform) but not by the less selective inhibitor N^G-nitro-L -arginine methylester (NAME). Western blotting experiments with monoclonal antibodies against the ncNOS and iNOS isoforms suggest that RA-elicited NOS activation is not attributable to an increased expression of the protein. NAME and AG were not able to revert inhibition of proliferation induced by RA, and the NO donor sodium nitroprusside did not mimic the effect of RA and PMA. These data indicate that increased NO synthesis does not mediate RA- or PMA-induced differentiation but may be an additional marker of differentiation into sympathetic-like neuronal cells. J. Cell. Physiol. 174:99–106, 1998. © 1998 Wiley-Liss, Inc.     

Nitric oxide induces BNIP3 expression that causes cell death in macrophages

Nitric oxide (NO) is involved in many physiological processes and also causes pathological effects by inducing apoptosis. It can enhance or suppress apoptosis depending on its concentration and the cell type involved. In this report, we used cDNA microarray analysis to show that SNAP, an NO donor, strongly induces Bcl-2/adenovirus E1B 19kDa-interacting protein 3 (BNIP3) in macrophages. BNIP3 is a mitochondrial pro-apoptotic protein that contains a Bcl-2 homology 3 domain and a COOH-terminal transmembrane (TM) domain. Macrophages activated by LPS/IFN-gamma produce nitric oxide synthase 2 (NOS2) and release endogenous NO. Expression of BNIP3 was also induced in macrophages by LPS/IFN-gamma, and the induction was blocked by a NOS2 inhibitor, S-methyl-isothiourea. Peritoneal macrophages from NOS2-null mice failed to produce BNIP3 in response to LPS/IFN-gamma. We conclude that BNIP3 expression in macrophages is controlled by the intracellular level of nitric oxide. Overexpression of BNIP3 but not of BNIP3 deltaTM, a BNIP3 mutant without the TM domain and C-terminal tail, led to apoptosis of the cells. Promoter analysis showed that the region between -281 and -1 of the 5'-upstream enhancer region of murine BNIP3 was sufficient for NO-dependent expression of BNIP3.     

NO signalling in cytokinin-induced programmed cell death

Cell death can be induced by cytokinin 6-benzylaminopurine (BA) at high dosage in suspension-cultured Arabidopsis cells. Herein, we provide evidence that BA induces nitric oxide (NO) synthesis in a dose-dependent manner. A reduction in cell death can be observed when the cytokinin is supplemented with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or the nitric oxide synthase (NOS) inhibitors: 2-aminoethyl-isothiourea (AET) and N^G.-monomethyl- l -arginine ( l -NMMA), which suggests that NO is produced via a NOS and is a signalling component of this form of programmed cell death. In BA-treated cells, mitochondrial functionality is altered via inhibition of respiration. This inhibition can be prevented by addition of either cPTIO or AET implying that NO acts at the mitochondrial level.