NF-kappaB Activation In Macrophages Research Articles

Abstract 578: Na/K-ATPase Forms a Signaling Complex with CD36 and Toll-like Receptor 4 to Mediate NF-kappaB Activation in Macrophages

Cardiotonic steroids such as ouabain, digoxin, and marinobufagenin are known ligands for the plasma membrane receptor, Na/K-ATPase (NKA). These ligands stimulate complex formation of NKA with other membrane and cytosolic proteins leading to cellular events such as activation of kinase cascades and gene transcription independent of the ion transporting function. Endogenous cardiotonic steroids have been detected within human circulation and their levels are elevated in patients with diseases associated with chronic systemic inflammation. However, the role of cardiotonic steroids and NKA-mediated signaling in inflammation has not been well studied. We now report that ouabain activates the NF-κB pathway in human monocyte-derived macrophages, leading to pro-inflammatory responses. Using Western blot and densitometry analysis, we found that physiological concentrations of ouabain stimulated IκBα degradation (n=3). Using quantitative RT-PCR (n=4), we found that ouabain increased mRNA levels of pro-inflammatory genes MCP-1 (3.2±1.1 fold), TNF-α (59.7±35.6 fold), CXCL-10 (2.8±1.6 fold), ICAM1 (2.9±0.5 fold), and PTGS2 (3.1±0.1 fold). Consistent with the increase in mRNA level, MCP-1 protein levels in post-culture media assessed by ELISA were elevated by 1.4±0.1 fold (n=4). Mechanistically, when macrophages were incubated with ouabain for 20 min and then subjected to IP, we found that antibodies to NKA co-precipitated more TLR4 (2.2±0.8 fold) compared to unstimulated cells. Additionally, antibodies to CD36 co-precipitated more NKA (1.8±0.6 fold) and more TLR4 (2.2±0.5 fold) (n=3). Blockade of TLR4 signaling using a specific inhibitor, CLI-095, abolished ouabain-induced MCP-1 production as assessed by ELISA (n=4). Next, we used siRNA to specifically knock down either NKA α1, CD36, TLR4 or MyD88 (the adaptor protein downstream of TLR4) in THP-1 cells stably transfected with a NF-κB reporter construct. After siRNA transfection for 48h, cells were treated with ouabain for additional 24h. Knockdown of any one of these proteins fully suppressed ouabain-induced NF-κB activation by NF-κB reporter assay (n=4). In conclusion, ouabain activates NF-κB through an NKA/CD36/TLR4 complex leading to pro-inflammatory responses in human macrophages.

Practical strategies for modulating foam cell formation and behavior.

Although high density lipoprotein (HDL)-mediated reverse cholesterol transport is crucial to the prevention and reversal of atheroma, a recent meta-analysis makes evident that current pharmaceutical strategies for modulating HDL cholesterol levels lower cardiovascular risk only to the extent that they concurrently decrease low density lipoprotein (LDL) cholesterol. This corresponds well with findings of a recent Mendelian randomization analysis, in which genetic polymorphisms associated with HDL cholesterol but no other known cardiovascular risk factors failed to predict risk for myocardial infarction. Although it is still seems appropriate to search for therapies that could improve the efficiency with which HDL particles induce reverse cholesterol transport, targeting HDL cholesterol levels per se with current measures appears to be futile. It may therefore be more promising to promote reverse cholesterol transport with agents that directly target foam cells. Macrophage expression of the cholesterol transport proteins adenosine triphosphate binding cassette transporter A1, adenosine triphosphate binding cassette transporter G1, and scavenger receptor class B member 1 is transcriptionally up-regulated by activated liver X receptors (LXR), whereas nuclear factor (NF)-kappaB antagonizes their expression. Taurine, which inhibits atherogenesis in rodent studies, has just been discovered to act as a weak agonist for LXRalpha. Conversely, it may be possible to oppose NF-kappaB activation in macrophages with a range of measures. Induction of heme oxygenase-1, which can be attained with phase 2 inducer phytochemicals such as lipoic acid and green tea catechins, promotes reverse cholesterol transport in macrophages and inhibits atherogenesis in rodents, likely due to, in large part, NF-kappaB antagonism. Inhibition of macrophage nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity with the spirulina-derived bilirubin-mimetic phycocyanobilin may also oppose NF-kappaB activation, and salicylic acid similarly should be useful for this purpose. The 5' adenosine monophosphate-activated protein kinase activator berberine promotes macrophage reverse cholesterol transport in cell culture; metformin probably shares this property. Many of these measures could also be expected to promote plaque stability by suppressing foam cell production of inflammatory cytokines and matrix metalloproteinases, and to reduce intimal monocyte infiltration by anti-inflammatory effects on vascular endothelium. Direct targeting of foam cells with agents such as phase 2 inducers, spirulina, salicylate, taurine, and berberine or metformin, may hence have considerable potential for preventing and reversing atheroma, and for preventing the plaque rupture that triggers vascular thrombosis.

Abstract 2841: Inducible modulation of NF-kappaB in macrophages reveals that timing of intervention may be critical to therapeutic outcome during mammary tumorigenesis

Abstract Elevated levels of NF-kappaB signaling are associated with multiple cancers. Therefore, it is not surprising that inhibitors of NF-kappaB are being investigated as potential therapeutics. While extensive efforts are being made to identify inhibitors there is less focus on investigating the effects of inhibition in specific cell types and during defined stages of tumor progression. We have developed murine transgenic models that enable us to induce expression of an activator or inhibitor of NF-kappaB in macrophages, by adding doxycycline to the drinking water of mice. We have combined these with the Polyoma model of mammary cancer to investigate the effects of modulation of NF-kappaB signaling specifically within macrophages. Given several recent publications we were predicting that activation of NF-kappaB would have pro-tumor effects. However, using a tail vein injection model, our data identifies a stage during progression in which activation of NF-kappaB in macrophages significantly limits metastasis. Activation of NF-kappaB alters immune cell populations and results in rapid killing of tumor cells during the seeding phase. In this model inhibition of NF-kappaB in macrophages during this critical stage of metastasis is pro-tumor. This suggests that while inhibition of NF-kappaB at later stages of tumor progression may have therapeutic benefit, inhibition during the seeding phase my lead to the opposite effect. Our studies highlight the importance of continued investigation into aspects of cell specificity and timing that may change the potential outcome of a clinical trial using NF-kappaB inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2841. doi:10.1158/1538-7445.AM2011-2841

Abstract 1352: Macrophage specific regulation reveals both pro- and anti-tumor effects of NF-kappaB during mammary tumor progression

Abstract An increased level of macrophages in human tumors is associated with a poorer prognosis. The transcription factor Nuclear Factor-kappaB (NF-kappaB) is an important regulator of gene expression within macrophages. Recent studies point to a pro-tumorigenic role for NF-kappaB activity in macrophages; however NF-kappaB also regulates expression of genes such as inducible nitric oxide synthase which are associated with anti-tumor effects. We believe that NF-kappaB activity in macrophages can have both anti- and pro-tumor effects depending on tumor stage. We have developed novel murine models to determine the effects of modulation of NF-kappaB activity specifically within macrophages during primary mammary tumor growth and metastasis. We have used the cfms promoter to specifically express the reverse tetracycline transactivator (rtTA) protein in macrophages. The rtTA can drive tet operon regulated gene expression in the presence of doxycyline (dox). We have used this with a dominant negative IkappaBalpha to inhibit NF-kappaB signaling in the Polyoma mouse mammary tumor model, this model is termed DNFP. We have also used the cfms-rtTA in combination with a constitutively active IKK2 transgene to activate NF-kappaB signaling, this model is termed IKFM. When NF-kappaB is inhibited in macrophages during primary tumor growth in our DNFP model there is a reduction in primary tumor growth accompanied by a reduced level of lung metastasis. This data suggests that NF-kappaB in macrophages is acting in a pro-tumor manner, in agreement with recently published data. In contrast, we have found that activation of NF-kappaB in macrophages by treating IKFM mice with dox for 1 week before and during two weeks following tail vein injection of polyoma mammary tumor cells leads to a significant reduction in surface lung tumor formation. In additional studies, IKFM mice were pretreated with dox for 1 week but treatment was stopped two days after tumor injection. A similar reduction in tumor number was observed, suggesting an inhibition of cell seeding. Real time PCR analysis of lungs from mice with activation of NF-kappaB in macrophages suggests an anti-tumor “M1” macrophage phenotype. In summary, our data indicates that NF-kappaB activity in macrophages can have both anti- and pro-tumor effects dependent on tumor stage. This data would suggest that cell specificity and timing of treatment are important considerations with regards to the use of NF-kappaB inhibitors in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1352.

Novel Anti-Inflammatory Action of 5-Aminoimidazole-4-carboxamide Ribonucleoside with Protective Effect in Dextran Sulfate Sodium-Induced Acute and Chronic Colitis

AMP-activated protein kinase (AMPK) is an important cellular energy sensor that is responsible for maintaining systemic and cellular energy balance. Its role in intestinal inflammation remains unclear. Recent studies indicate that AMPK activation initiated by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) participates in modulating inflammatory responses. Inflammatory bowel disease (IBD) has been characterized by sustained intestinal mucosa inflammation, caused mainly by excessive macrophage activation and T helper type 1 (Th1) and Th17 immune responses. Thus, we sought to determine the effect of AICAR on inflammatory responses of murine models of IBD. Mice with acute or chronic colitis induced by dextran sulfate sodium (DSS) were treated with or without AICAR. Body weight and colon inflammation were evaluated, and production of proinflammatory cytokines in colon tissues was determined. Nuclear factor kappaB (NF-kappaB) activation in colon tissues was assayed, and Th1 and Th17 cell responses were also evaluated. By inducing AMPK activation, AICAR had a therapeutic effect in ameliorating acute and chronic DSS-induced murine colitis as shown by reduced body weight, loss and significant attenuation in clinical symptoms, and histological inflammation. Moreover, AICAR treatment inhibited NF-kappaB activation in macrophages, reduced levels of Th1- and Th17-type cytokines in colon tissues, and down-regulated Th1 and Th17 cell responses during the progress of acute and chronic experimental colitis. AICAR acts as a central inhibitor in immune responses of experimental colitis. Our data show that AICAR-initiated AMPK activation may represent a promising alternative to our current approaches to suppress intestinal inflammation in IBD.

Modulation of the NF-κB Pathway by Bordetella pertussis Filamentous Hemagglutinin

BackgroundFilamentous hemagglutinin (FHA) is a cell-associated and secreted adhesin produced by Bordetella pertussis with pro-apoptotic and pro-inflammatory activity in host cells. Given the importance of the NF-κB transcription factor family in these host cell responses, we examined the effect of FHA on NF-κB activation in macrophages and bronchial epithelial cells, both of which are relevant cell types during natural infection.Methodology/Principal FindingsExposure to FHA of primary human monocytes and transformed U-937 macrophages, but not BEAS-2B epithelial cells, resulted in early activation of the NF-κB pathway, as manifested by the degradation of cytosolic IκBα, by NF-κB DNA binding, and by the subsequent secretion of NF-κB-regulated inflammatory cytokines. However, exposure of macrophages and human monocytes to FHA for two hours or more resulted in the accumulation of cytosolic IκBα, and the failure of TNF-α to activate NF-κB. Proteasome activity was attenuated following exposure of cells to FHA for 2 hours, as was the nuclear translocation of RelA in BEAS-2B cells.ConclusionsThese results reveal a complex temporal dynamic, and suggest that despite short term effects to the contrary, longer exposures of host cells to this secreted adhesin may block NF-κB activation, and perhaps lead to a compromised immune response to this bacterial pathogen.

Evidence for a Pathogenic Determinant in HIV-1 Nef Involved in B Cell Dysfunction in HIV/AIDS

B lymphocyte hyperactivation and elevated immunoglobulin levels (hypergammaglobulinemia) are pathogenic manifestations of HIV-1 infection. Here we provide evidence that these hallmarks are caused by a soluble factor whose production by infected macrophages is induced by the HIV-1 Nef protein. In vitro, HIV-1-infected macrophages or macrophages expressing Nef promoted B cell activation and differentiation to immunoglobulin-secreting cells. Nef-mediated activation of NF-kappaB in macrophages induced secretion of the acute-phase protein ferritin, and ferritin was necessary and sufficient for the observed Nef-dependent B cell changes. The extent of hypergammaglobulinemia in HIV-1-infected individuals correlated directly with plasma ferritin levels and with viral load. Furthermore, the induction of ferritin production and hypergammaglobulinemia was recapitulated when Nef was specifically expressed in macrophages and T cells of transgenic mice. Collectively, these results indicate that the HIV-1 Nef protein carries a pathogenic determinant that governs B cell defects in HIV-1 infection.

Water solution of onion crude powder inhibits RANKL-induced osteoclastogenesis through ERK, p38 and NF-κB pathways

Onion powder has been reported to decrease the ovariectomy-induced bone resorption of rats. However, the molecular mechanism of onion powder on the bone cells has not been reported. Here, we report that water solution of onion crude powder decreases the osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells. Additionally, water solution of onion crude powder inhibits the RANKL-induced ERK, p38 and NF-kappaB activation in macrophages. In summary, our data showed that onion powder may benefit bone through an anti-resorption effect on the osteoclasts. A nutritional approach is important for both prevention and treatment of osteoporosis. Onion has been reported to decrease the ovariectomy-induced bone resorption. However, the functional effects of onion on the cultured osteoclasts and osteoblasts remain largely unknown. Here, we found that water solution of onion crude powder markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis through ERK, p38 and NF-kappaB pathways. Other studies were also designed to investigate the potential signaling pathways involved in onion-induced decrease in osteoclastogenesis. The osteoclastogenesis was examined using the TRAP staining method. The MAPKs and NF-kappaB pathways were measured using Western blot analysis. A transfection protocol was used to examine NF-kappaB activity. Water solution of onion crude powder inhibited the RANKL plus M-CSF-induced osteoclastic differentiation from either bone marrow stromal cells or from RAW264.7 macrophage cells. Treatment of RAW264.7 macrophages with RANKL could induce the activation of ERK, p38 and NF-kappaB that was inhibited by water solution of onion crude powder. On the other hand, it did not affect the cell proliferation and differentiation of human cultured osteoblasts. Our data suggest that water solution of onion crude powder inhibits osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells via attenuation of RANKL-induced ERK, p38 and NF-kappaB activation.

Anti‐inflammatory effects of liquiritigenin as a consequence of the inhibition of NF‐κB‐dependent iNOS and proinflammatory cytokines production

Glycyrrhizae radix has been widely used as a cytoprotective, plant-derived medicine. We have identified a flavanoid, liquiritigenin, as an active component in extracts of Glycyrrhizae radix. This research investigated the effects of liquiritigenin on the induction of inducible NOS (iNOS) and proinflammatory cytokines by lipopolysaccharide (LPS) in Raw264.7 cells, and on paw oedema in rats. iNOS expression was determined by western blotting, real-time reverse transcription-PCR and reporter gene analyses. Tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and IL-6 were assayed by ELISA. Gel shift assay and immunoblotting were used to assess NF-kappaB activation. The effect of liquiritigenin on acute inflammation in vivo was evaluated using carrageenan-induced paw oedema. Treatment of Raw264.7 cells with liquiritigenin caused inhibition of LPS-induced NF-kappaB DNA binding activity, due to repression of I-kappaBalpha phosphorylation and degradation. Liquiritigenin treatment prevented LPS from increasing the levels of iNOS protein and mRNA in a concentration-dependent manner. Liquiritigenin also suppressed the production of TNF-alpha, IL-1beta and IL-6 from Raw264.7 cells after LPS. In rats, liquiritigenin treatment inhibited formation of paw oedema induced by carrageenan. These results demonstrate that liquiritigenin exerts anti-inflammatory effects, which results from the inhibition of NF-kappaB activation in macrophages, thereby decreasing production of iNOS and proinflammatory cytokines. Our findings showing inhibition by liquiritigenin of paw oedema as well as inflammatory gene induction will help to understand the pharmacology and mode of action of liquiritigenin, and of the anti-inflammatory use of Glycyrrhizae radix.

Plant sterol or stanol esters retard lesion formation in LDL receptor-deficient mice independent of changes in serum plant sterols

Statins do not always decrease coronary heart disease mortality, which was speculated based on increased serum plant sterols observed during statin treatment. To evaluate plant sterol atherogenicity, we fed low density lipoprotein-receptor deficient (LDLr(+/-)) mice for 35 weeks with Western diets (control) alone or enriched with atorvastatin or atorvastatin plus plant sterols or stanols. Atorvastatin decreased serum cholesterol by 22% and lesion area by 57%. Adding plant sterols or stanols to atorvastatin decreased serum cholesterol by 39% and 41%. Cholesterol-standardized serum plant sterol concentrations increased by 4- to 11-fold during sterol plus atorvastatin treatment versus stanol plus atorvastatin treatment. However, lesion size decreased similarly in the sterol plus atorvastatin (-99% vs. control) and the stanol plus atorvastatin (-98%) groups, with comparable serum cholesterol levels, suggesting that increased plant sterol concentrations are not atherogenic. Our second study confirms this conclusion. Compared with lesions after a 33 week atherogenic period, lesion size further increased in controls (+97%) during 12 more weeks on the diet, whereas 12 weeks with the addition of plant sterols or stanols decreased lesion size (66% and 64%). These findings indicate that in LDLr(+/-) mice 1) increased cholesterol-standardized serum plant sterol concentrations are not atherogenic, 2) adding plant sterols/stanols to atorvastatin further inhibits lesion formation, and 3) plant sterols/stanols inhibit the progression or even induce the regression of existing lesions.

Cardiac glycoside inhibits IL‐8‐induced biological responses by downregulating IL‐8 receptors through altering membrane fluidity

Considering the potential role of interleukin-8 (IL-8) in inflammation, angiogenesis, tumorogenesis, and metastasis, and the involvement of different cell types especially neutrophils and macrophages in those processes, the regulation of IL-8-mediated biological responses is important. In this report we provide evidences that oleandrin, a cardiac glycoside potentially inhibited IL-8-, formyl peptide (FMLP)-, EGF-, or nerve growth factor (NGF)-, but not IL-1- or TNF-induced NF-kappaB activation in macrophages. Oleandrin inhibited IL-8-, but not TNF-induced NF-kappaB-dependent genes expression. Oleandrin inhibited the binding of IL-8, EGF, or NGF, but not IL-1 or TNF. It decreased almost 79% IL-8 binding without altering affinity towards IL-8 receptors and this inhibition of IL-8 binding was observed in isolated membrane. The IL-8, anti-IL-8Rs antibodies, or protease inhibitors were unable to protect oleandrin-mediated inhibition of IL-8 binding. Phospholipids significantly protected oleandrin-mediated inhibition of IL-8 binding thereby restoring IL-8-induced NF-kappaB activation. Oleandrin altered the membrane fluidity as detected by microviscosity parameter and a decrease in diphenylhexatriene, a lipid binding fluorophore binding in a dose-dependent manner. Overall, our results suggest that oleandrin inhibits IL-8-mediated biological responses in diverse cell types by modulating IL-8Rs through altering membrane fluidity and microviscosity. The study might help to regulate IL-8-mediated biological responses involved in inflammation, metastasis, and neovascularization.

Macrophage-specific overexpression of group IIa sPLA2 increases atherosclerosis and enhances collagen deposition

Atherosclerosis is a chronic inflammatory disease of the vessel wall characterized by the accumulation of lipid-laden macrophages and fibrotic material. The initiation of the disease is accompanied by the accumulation of modified lipoproteins in the vessel wall. Group IIa secretory phospholipase A2 (sPLA2 IIa) is a key candidate player in the enzymatic modification of low density lipoproteins. To study the role of sPLA2 IIa in macrophages during atherogenesis, transgenic mice were generated using the human sPLA2 IIa gene and the CD11b promoter. Bone marrow transplantation to LDL receptor-deficient mice was performed to study sPLA2 IIa in atherosclerosis. After 10 weeks of high-fat diet, mice overexpressing sPLA2 IIa in macrophages showed 2.3-fold larger lesions compared with control mice. Pathological examination revealed that sPLA2 IIa-expressing mice had increased collagen in their lesions, independent of lesion size. However, smooth muscle cells or fibroblasts in the lesions were not affected. Other parameters studied, including T-cells and cell turnover, were not significantly affected by overexpression of sPLA2 IIa in macrophages. These data clearly show that macrophage sPLA2 IIa is a proatherogenic factor and suggest that the enzyme regulates collagen production in the plaque and thus fibrotic cap development.

Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice.

Atherosclerosis is now generally accepted as a chronic inflammatory condition. The transcription factor NF-kappaB is a key regulator of inflammation, immune responses, cell survival, and cell proliferation. To investigate the role of NF-kappaB activation in macrophages during atherogenesis, we used LDL receptor-deficient mice with a macrophage-restricted deletion of IkappaB kinase 2 (IKK2), which is essential for NF-kappaB activation by proinflammatory signals. These mice showed increased atherosclerosis as quantified by lesion area measurements. In addition, the lesions were more advanced and showed more necrosis and increased cell number in early lesions. Southern blotting revealed that deletion of IKK2 was approximately 65% in macrophages, coinciding with a reduction of 50% in NF-kappaB activation, as compared with controls. In both groups, the expression of differentiation markers, uptake of bacteria, and endocytosis of modified LDL was similar. Upon stimulation with LPS, production of TNF was reduced by approximately 50% in IKK2-deleted macrophages. Interestingly, we also found a major reduction in the anti-inflammatory cytokine IL-10. Our data show that inhibition of the NF-kappaB pathway in macrophages leads to more severe atherosclerosis in mice, possibly by affecting the pro- and anti-inflammatory balance that controls the development of atherosclerosis.

Silica-induced apoptosis in murine macrophage: involvement of tumor necrosis factor-alpha and nuclear factor-kappaB activation.

Alveolar macrophages play a critical role in silica-induced lung fibrosis. Silica exposure induces tumor necrosis factor (TNF)-alpha release and nuclear factor (NF)-kappaB activation, and apoptotic mechanisms have been implicated in silica-induced pathogenesis. To characterize potential relationships between these signaling events, we studied their induction in two murine macrophage cell lines. The RAW 264.7 macrophage cell line was more sensitive, and the IC-21 macrophage cell line more tolerant to silica exposure (0.2 or 1 mg/ml for 6 h) as evidenced by significantly higher apoptotic responses in RAW 264.7 (P < 0.05). RAW 264.7 macrophages exhibited enhanced TNF-alpha production and NF-kappaB activation in response to silica, whereas IC-21 macrophages did not produce TNF-alpha in response to silica and did not induce NF-kappaB nuclear binding. Inhibition of NF-kappaB in RAW 264.7 cells with BAY11-7082 significantly increased apoptosis while inhibiting TNF-alpha release. In addition, TNF-alpha and NF-kappaB activation, but not apoptosis, were induced by lipopolysaccharide (LPS) in both cell lines, and NF-kappaB inhibition reduced LPS-induced TNF-alpha release. These data suggest that TNF-alpha induction is dependent on NF-kappaB activation in both cell lines. However, silica can induce apoptosis in murine macrophages, independently of TNF-alpha stimulation, as in IC-21 macrophages. Furthermore, NF-kappaB activation in macrophages may play dual roles, both pro- and antiapoptotic during silica injury.

Nitric oxide up-regulates DNA-binding activity of nuclear factor-kappaB in macrophages stimulated with silica and inflammatory stimulants.

Nitric oxide (NO), a reactive nitrogen species, plays an important role in inflammatory lung damage. In the present study, we investigated the role of NO in DNA-binding activity of NF-kappaB in macrophages stimulated with silica or other inflammatory stimulants. Treatment of mouse macrophages (RAW264.7 cells) with a selective inhibitor of inducible nitric oxide synthase (iNOS), L-N6-(1-iminoethyl) lysine (L-NIL), or a nonselective iNOS inhibitor, N omega-nitro-L-arginine methylester (L-NAME), resulted in inhibition of silica-induced nitric oxide production as well as silica-induced NF-kappaB activation. L-NIL also effectively inhibited NF-kappaB activation induced by other inflammatory stimulants, such as lipopolysaccharide (LPS) or muramyl dipeptide (MDP). These inhibitory effects of L-NIL and L-NAME on silica- or LPS-induced NF-kappaB activation were also observed in primary rat alveolar macrophages. Furthermore, NO generating compounds, such as sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), caused a dose-dependent increase in NF-kappaB activation, which was positively correlated with the level of NO production. Specific inhibitors of protein tyrosine kinase, such as genistein and AG494, prevented NF-kappaB activation in SNP- or SIN-1 treated cells, suggesting involvement of tyrosine kinase in the NO signaling pathway leading to NF-kappaB activation. In contrast, inhibitors of protein kinase C or A, such as staurosporine or H89, had no inhibitory effect on SIN-1 induced NF-kappaB activation. Metalloporphyrins, such as tetrakis (N-methyl-4'-pyridyl) porphyrinato iron (III) (Fe-TMPyP) and Zn-TMPyP which are known to alter NO-dependent activity, markedly inhibited silica- and LPS-induced NF-kappaB activation. The results suggest that NF-kappaB activation in macrophages can be induced under certain conditions by nitric oxide and that nitric oxide produced by phagocytes exposed to inflammatory agents may up-regulate the activation of NF-kappaB.

Resistance to endotoxic shock as a consequence of defective NF-kappaB activation in poly (ADP-ribose) polymerase-1 deficient mice.

Poly (ADP-ribose) polymerase-1 is a nuclear DNA-binding protein that participates in the DNA base excision repair pathway in response to genotoxic stress in mammalian cells. Here we show that PARP-1-deficient cells are defective in NF-kappaB-dependent transcription activation, but not in its nuclear translocation, in response to TNF-alpha. Treating mice with lipopolysaccharide (LPS) resulted in the rapid activation of NF-kappaB in macrophages from PARP-1(+/+) but not from PARP-1(-/-) mice. PARP-1-deficient mice were extremely resistant to LPS-induced endotoxic shock. The molecular basis for this resistance relies on an almost complete abrogation of NF-kappaB-dependent accumulation of TNF-alpha in the serum and a down-regulation of inducible nitric oxide synthase (iNOS), leading to decreased NO synthesis, which is the main source of free radical generation during inflammation. These results demonstrate a functional association in vivo between PARP-1 and NF-kappaB, with consequences for the transcriptional activation of NF-kappaB and a systemic inflammatory process.

The role of hydroxyl radical as a messenger in the activation of nuclear transcription factor NF-kappaB.

Although it is generally believed that reactive oxygen species activate NF-kappaB, a primary oxidative stress-responsive transcription factor, it is unclear which one among these species causes NF-kappaB activation. Our hypothesis is that hydroxyl radical (*OH) functions as a messenger for the activation of NF-kappaB. Jurkat cells, macrophages and JB6 cells were used to test this hypothesis. Cr(VI), silica and ZnO were used as sources of *OH radicals. None of these *OH generating systems involves exogenous H2O2. Cr(VI) expressed enhanced activity in induction of NF-kappaB in Jurkat cells. This activation of NF-kappaB was decreased by a metal chelator, diethylene triaminepentaacetic acid or a H2O2 scavenger, catalase, but was increased by superoxide dismutase. Mn(II), which reacts with Cr(IV) to inhibit this metal ion-mediated *OH generation, decreased the NF-kappaB activation. Sodium formate, an *OH radical scavenger, also inhibited the NF-kappaB activation. Electron spin resonance measurements show that Cr(VI) was reduced by Jurket cells to Cr(IV) and Cr(V). During the reduction process, molecular oxygen was reduced to O2 and then to H2O2, which reacted with Cr(IV) and Cr(V) to generate *OH radical. The *OH generation correlated with the Cr(VI)-induced NF-kappaB activation. Similarly, silica caused NF-kappaB activation in macrophages via the *OH radical-mediated reaction. This radical was generated via metal mediated reaction from H2O2, which was generated by the reduction of molecular oxygen via O2- as an intermediate during the silica-stimulated 'respirable burst'. Silica particles did not cause *OH generation either in Jurket or in JB6 cells and thus did not cause any observable NF-kappaB activation in these cells. ZnO induced NF-kappaB activation in JB6 cells through the generation of *OH resulting from light irradiation of ZnO which was measured by electron spin resonance. The results thus show that *OH radical functions as a messenger for NF-kappaB activation. Antioxidants, which scavenge *OH radical or its precursors, inhibit NF-kappaB activation. Metal chelators, which make metal ions incapable of generating *OH from H2O2, inhibit activation of this transcription factor.