Murine Peritoneal Macrophages Research Articles

The inhibition of JNK MAPK and NF-κB signaling by tenuifoliside A isolated from Polygala tenuifolia in lipopolysaccharide-induced macrophages is associated with its anti-inflammatory effect

The root of Polygala tenuifolia Willd. (Polygalaceae) is well known for its use in the treatment of neurasthenia, amnesia, and inflammation. In this study, we isolated phenyl propanoid type metabolite tenuifoliside A, one of the phenylpropanoids from P. tenuifolia, and investigated its anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated RAW264.7 and murine peritoneal macrophages. The results showed that tenuifoliside A inhibited the production of nitric oxide (NO), inducible nitric oxide synthase (iNOS), prostaglandin E2 (PG E2), and cyclooxygenase (COX)-2. In addition, tenuifoliside A suppressed the production of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β. We also evaluated the effects of tenuifoliside A on the activation of nuclear factor-kappaB (NF-κB). Tenuifoliside A inhibited the translocation of the NF-κB subunit p65 into the nucleus by interrupting the phosphorylation and degradation of inhibitor kappa B (IκB)-α in LPS-stimulated murine peritoneal macrophages. Moreover, we confirmed that the suppression of the inflammatory process by tenuifoliside A was mediated through the mitogen-activated protein kinases (MAPKs) pathway based on the fact that tenuifoliside A significantly decreased p-c-Jun N-terminal kinase (p-JNK) protein expression in LPS-stimulated murine peritoneal macrophages. Taken together, the anti-inflammatory effects of tenuifoliside A were mediated by the inhibition of the NF-κB and MAPK pathways. This study is the first report on the anti-inflammatory effects of tenuifoliside A, and the strong anti-inflammatory effects of tenuifoliside A provide potential compound to be developed as therapeutic for inflammatory diseases.

Salt-Dependent Chemotaxis of Macrophages

Besides their role in immune system host defense, there is growing evidence that macrophages may also be important regulators of salt homeostasis and blood pressure by a TonEBP-VEGF-C dependent buffering mechanism. As macrophages are known to accumulate in the skin of rats fed under high salt diet conditions and are pivotal for removal of high salt storage, the question arose how macrophages sense sites of high sodium storage. Interestingly, we observed that macrophage-like RAW264.7 cells, murine bone marrow-derived macrophages and peritoneal macrophages recognize NaCl hypertonicity as a chemotactic stimulus and migrate in the direction of excess salt concentration by using an in vitro transwell migration assay. While RAW264.7 cells migrated toward NaCl in a dose-dependent fashion, no migratory response toward isotonic or hypotonic media controls, or other osmo-active agents, e.g. urea or mannitol, could be detected. Interestingly, we could not establish a specific role of the osmoprotective transcription factor TonEBP in regulating salt-dependent chemotaxis, since the specific migration of bone marrow-derived macrophages following RNAi of TonEBP toward NaCl was not altered. Although the underlying mechanism remains unidentified, these data point to a thus far unappreciated role for NaCl-dependent chemotaxis of macrophages in the clearance of excess salt, and suggest the existence of novel NaCl sensor/effector circuits, which are independent of the TonEBP system.

Antineoplastic drug NSC631570 modulates functions of hypoxic macrophages

Hypoxia is an important factor in the macrophages microenvironment. Many physiological and pathological processes including solid tumor development are characterized by both low oxygen content and presence of macrophages. Tumor-associated hypoxia causes alternative polarization of macrophages in tumor tissue and transformation of these cells into the allies of a malignant neoplasm. The aim of the work was to investigate the effect of NSC631570, a cancer-selective drug that is known to selectively accumulate in the tumor tissue, on hypoxic macrophage function. Murine peritoneal macrophages (PMs) were subjected to hypoxia (3% O2). Nitrite level was assayed by the Griess reaction. Arginase activity was measured by colorimetric method. ROS generation and phagocytosis was estimated by flow cytometry. O2(-) generation was assayed by the NBT reduction method. HMGB1 expression was determined by ELISA. 42 h hypoxia caused alternative polarization of murine PMs with significant arginase prevalence. NSC631570 repolarized arginine metabolism of hypoxic macrophages to NOS dominant and activated their pro-inflammatory functions: recovered ROS production and increased alarmin release NSC631570 can restore pro-inflammatory functions of macrophages, alternatively polarized by hypoxia.

Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages

Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages.

Abstract 276: Shockwave Reduces Macrophage Foam Cell Formation Via Increased Expression of ATP Binding Cassette Transporters.

Background: Excessive lipid accumulation by macrophages plays a crucial role in atherosclerosis. Foam cells are generated by uncontrolled uptake of modified LDL, especially oxidized LDL (oxLDL), and/or impaired cholesterol efflux mediated by ATP-binding cassette (ABC) family transporters, ABCA-1 and ABCG-1. Shockwave, elicited by transient pressure disturbance, have been used for extracorporeal lithotripsy or for treating musculoskeletal disorders. Our current study suggests an evidence that shockwave may have anti-atherogenic effect by inhibiting foam cell formation. Methods/Results: Murine peritoneal macrophages were exposed to shockwaves at 0.04 mJ/mm 2 with 1000 impulses, lysed after 6, 18 and 24 hours, and tested for expression of ABCA-1 and ABCG-1. The western blot showed that shockwave induced 2.0-2.8 fold increase of ABCA-1 and ABCG-1 within 18-24 hours. mRNA levels of ABCA-1 and ABCG-1 were also increased by shockwave with 2.0 fold of peak increase in 18 hours. The increased expression of ABCA-1 and ABCG-1 was mediated by phosphorylation of ERK 1/2 (Tyr204). Western blot analysis revealed that shockwave induced phosphorylation of ERK 1/2 (Tyr204) in murine macrophages. Shockwave-induced increase of ABCA-1 and ABCG-1 was blocked by U0126 (40µM), a specific inhibitor for ERK. Oil-red O staining showed that macrophages exposed to shockwave had 25% less intracellular lipid droplets. Intracellular cholesterol measured by cholesterol oxidase and esterase revealed that macrophages exposed to shockwave had 23% less intracellular cholesterol when incubated with oxLDL (50µg/ml) for 16 hours. In vitro migration assays including modified Boyden chamber migration assay and scratch wound healing migration assay showed that macrophages exposed to shockwave had 1.2 fold more migration and had diminished migration-inhibitory effect of oxLDL. Conclusions: Shockwave reduces macrophage foam cell formation via ERK-mediated increase of ABCA-1 and ABCG-1 mediating lipid efflux and promotes macrophage migration which may induce macrophage egress from atherosclerotic lesion. Our study suggests anti-atherogenic effects of shockwave as a potential treatment modality for atherosclerosis.

Bioenergetic Profiles Diverge During Macrophage Polarization: Implications for the Interpretation of 18F-FDG PET Imaging of Atherosclerosis

Conventional cardiovascular imaging is invaluable for the assessment of late sequelae of atherosclerosis, such as diminished perfusion reserve and luminal stenosis. Molecular imaging provides complementary information about plaque composition and ongoing biologic processes in the vessel wall, allowing the early diagnosis and risk stratification of patients. Detection of enhanced glucose uptake, using (18)F-FDG PET, has been proposed as a noninvasive approach to track macrophage activation as a critical event in the development and progression of atherosclerosis. In this study, we determined the impact of macrophage polarization on glucose metabolism and oxidative phosphorylation. Murine peritoneal macrophages were incubated in the presence of interferon-γ (IFN-γ) plus tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS), or interleukin-4 (IL-4) to induce classic (M1 and M(LPS)) or alternative (M2) polarization, respectively. Glucose uptake was measured using (3)H-deoxyglucose. Oxidative phosphorylation was evaluated using an extracellular flux analyzer. Mitochondrial DNA copy numbers were quantified by polymerase chain reaction. The expression of glucose transporter-1 (Glut-1), hexokinase-1 and -2 (Hk-1 and Hk-2, respectively), mitochondrial transcription factor-1 (Tfam), and cytochrome c oxidase subunit I (Cox-1) was determined by quantitative reverse transcription polymerase chain reaction. Stimulation of macrophages by LPS, but not polarization with either IFN-γ plus TNF-α (M1) or IL-4 (M2), resulted in a 2.5-fold increase in (3)H-deoxyglucose uptake. Enhanced glucose uptake by M(LPS) macrophages paralleled the overexpression of rate-limiting proteins involved in transmembrane transport and intracellular trapping of glucose--that is, Glut-1, Hk-1, and Hk-2. Alternatively polarized M2 macrophages developed a markedly higher spare respiratory capacity than both nonpolarized and classically polarized M1 macrophages. M2 polarization was associated with a 4.6-fold increase in mitochondrial content of the cells, compared with nonpolarized macrophages. The expression of Tfam, a major regulator of mitochondrial biogenesis, and Cox-1, a critical component of respiratory chain, was significantly increased in M2 polarized macrophages. Polarization of macrophages induces distinct metabolic profiles with respect to glycolysis versus oxidative phosphorylation, with alternatively polarized macrophages shifting to mitochondria as their main source of adenosine triphosphate. Only M(LPS), but not M1 or M2 polarized macrophages, showed increased glucose uptake, suggesting that glucose metabolism is regulated independent of the polarization state and macrophage polarization may not be detectable by (18)F-FDG PET.

Anti-inflammatory effects of silkworm hemolymph on lipopolysaccharide-stimulated macrophages

Macrophages participate in several inflammatory pathologies such as sepsis and arthritis. We investigated the effect of silkworm hemolymph (SH) on the LPS-induced pro-inflammatory macrophages. SH inhibits LPS-induced nitric oxide (NO) production in RAW 264.7 cells and murine peritoneal macrophages. The decreased NO was reflected as a decreased amount of inducible nitric oxide synthase (iNOS) mRNA and protein. It was also found that SH inhibited pro-inflammatory cytokines, IL-1β, IL-6, and TNF-α production. To elucidate the mechanism by which SH inhibits NO production and iNOS expression, we investigated that SH suppressed IκB phosphorylation, which leads to the activation of NF-κB followed by degradation of IκB. This observation suggests that SH is a potential therapeutic modulator for inflammation-associated disorders.

Baicalein Selectively Induces Apoptosis in Activated Lymphocytes and Ameliorates Concanavalin A-Induced Hepatitis in Mice

BackgroundInsufficient apoptosis in activated lymphocytes contributes to the development of autoimmune hepatitis (AIH). Baicalein (BE), a flavonoid originally isolated from the root of Scutellaria baicalensis Georgi, possesses anti-inflammatory properties. However, whether BE can selectively induce apoptosis in activated lymphocytes and exert therapeutic effect on AIH has not been studied.Methodology/Principal FindingsThe pro-apoptotic properties of BE were evaluated in vitro on different types of immune cells, and in vivo effects of BE were examined in a murine model of Concanavalin A (Con A)-induced hepatitis. In vitro treatment with BE resulted in a higher increase in the level of apoptosis in Con A-stimulated murine splenocytes, Con A-stimulated CD3+ splenocytes, lipopolysaccharide (LPS)-stimulated CD19+ splenocytes, and phorbol 12-myristate 13-acetate/ionomycin-stimulated Jurkat T cells, compared with that in unstimulated naïve ones. Murine bone marrow-derived dentritic cells, peritoneal macrophages, and RAW264.7 cells, either stimulated with LPS or unstimulated, were all insensitive to the BE-induced apoptosis. BE treatment also led to a loss of mitochondrial membrane potential, an increase of cytochrome c release from mitochondria to the cytosol, a decrease in the ratio of Bcl-2/Bax, and activation of caspase-9,-3 in Con A-stimulated CD3+ splenocytes and LPS-stimulated CD19+ splenocytes, while showing no impact on Fas/FasL expressions and caspase-8 activation. In vivo administration of BE alleviated Con A-induced liver injury, suppressed serum level of TNF-α and IFN-γ, and reduced liver infiltration of mononuclear cells (MNCs). Furthermore, BE treatment increased the incidences of apoptosis in liver-infiltrating MNCs and splenocytes, as well as in CD3+ and CD19+ splenocytes. When liver MNCs and splenocytes from BE-treated mice were cultured in vitro for 24 h, they exhibited marked increase in apoptosis compared to vehicle-treated control.Conclusions/SignificanceThe present study demonstrates the ability of BE to promote apoptosis in activated lymphocytes through mitochondrial pathway and its potential use in the treatment of AIH.

Spermine oxidase is a regulator of macrophage host response to Helicobacter pylori: enhancement of antimicrobial nitric oxide generation by depletion of spermine

The gastric pathogen Helicobacter pylori causes peptic ulcer disease and gastric cancer. We have reported that in H. pylori-activated macrophages, nitric oxide (NO) derived from inducible NO synthase (iNOS) can kill the bacterium, iNOS protein expression is dependent on uptake of its substrate L-arginine (L-Arg), the polyamine spermine can inhibit iNOS translation by inhibiting L-Arg uptake, and inhibition of polyamine synthesis enhances NO-mediated bacterial killing. Because spermine oxidase (SMO), which back-converts spermine to spermidine, is induced in macrophages by H. pylori, we determined its role in iNOS-dependent host defense. SMO shRNA knockdown in RAW 264.7 murine macrophages resulted in a marked decrease in H. pylori-stimulated iNOS protein, but not mRNA expression, and a 90% reduction in NO levels; NO production was also inhibited in primary murine peritoneal macrophages with SMO knockdown. There was an increase in spermine levels after H. pylori stimulation that rapidly decreased, while SMO knockdown caused a greater increase in spermine that was sustained. With SMO knockdown, L-Arg uptake and killing of H. pylori by macrophages was prevented. The overexpression of SMO by transfection of an expression plasmid prevented the H. pylori-stimulated increase in spermine levels, and led to increased L-Arg uptake, iNOS protein expression and NO production, and H. pylori killing. In two human monocytic cell lines, U937 and THP-1, overexpression of SMO caused a significant enhancement of NO production with H. pylori stimulation. By depleting spermine, SMO can abrogate the inhibitory effect of polyamines on innate immune responses to H. pylori by enhancing antimicrobial NO production.

Morphological effects of autologous hsp70 on peritoneal macrophages in a murine T cell lymphoma

Heat shock protein 70 is highly conserved cytosolic protein which have important role in growth, development, and apoptosis. Hsp70 is well-known activator of macrophages and enhances the release of specific and non-specific effector molecules that have major role in tumor destruction and immunopotentiation of host. However, morphological effects of hsp 70 has not been carried out, therefore, morphological effects of hsp 70 on murine peritoneal macrophages were examined by light microscopy and scanning electron microscopy. Thioglycolate-induced peritoneal macrophages were prepared from BALB/c mice and cultured for 24 h in the presence of the hsp70. Tumor-associated macrophages treated with 10 μg/ml were varied in shape, mostly spindle shaped, i.e., stretched bidirectionally; surface ruffles were increased and their lamellipodia was prominent which suggest that hsp 70 treatment not only enhances the functional state of the peritoneal macrophages but also initiate immense morphological changes leading to increased endothelium adherence, increased antigen uptake, and increased migration to the inflammatory site.

EVALUATION OF ERYTHROCYTE MEMBRANE STABILIZING ACTIVITY, HAEMOLYTIC ACTIVITY AND CYTOTOXIC EFFECT OF THE AREAL TUBERS OF DIOSCOREA ALATA L OF NORTH-EASTERN REGION OF INDIA

D. alata is a staple food in many regio ns of the world and also extensively consumed in the North - Eastern Region of India. Medicinal properties of the tubers of D. alata are well established, but this is the first time that the possible toxicological evaluation of the areal tubers has been done . Hydro - methanolic extract of the areal tubers were prepared and used for all the experiments. EMSA was estimated at 562 nm against quercetin as standar d. The haemolytic activity was assayed at 540 nm against Triton - X as the standard. The cytotoxic effect of D. alata was studied on murine splenocytes, thymocytes, hepatocytes and peritoneal macrophage by MTT cytotoxic test. All the results were statistically analysed. The half maximal haemolytic concent ration for D. alata and triton - X were 1312.94 ± 180.15µg /ml and 189.24 ± 1.54 µg/ml , respectively . The half maximal inhibitory concentration of the D. alata was 216.79 ± 8.73 µg/ml for EMSA and 2905.66 ± 538.34 µg/ml, 3997.91 ± 1873.94 µg/ml, 5201.23 ± 226.38 µg/ml and 4284.62 ± 1256.16 µg/ml, respectively, f or MTT assay. The

Abarema cochliacarpos reduces LPS-induced inflammatory response in murine peritoneal macrophages regulating ROS-MAPK signal pathway

Ethnopharmacological relevanceAbarema cochliacarpos (Gomes) Barneby and Grimes (Fabaceae), known by the vulgar name of Babatenã, has been traditionally used in Northeast Brazil, as an anti-inflammatory remedy. Previous studies have demonstrated its anti-inflammatory and antiulcer effects in skin lesion, alcohol gastric ulcer and acute and chronic colitis. AimsThe present study was designed to evaluate the antioxidant and anti-inflammatory effects of the butanolic fraction from A. cochliacarpos (BFAC) and its major flavonoid, (+)-catechin, in LPS-stimulated murine peritoneal macrophages. Moreover, we studied the role of mitogen-activated protein kinase (MAPK)s and NF-kB signaling pathways possibly involved in the beneficial effects. Materials and methodsThe quantification of the extract was carried out by ultra-performance liquid chromatography analysis. Cell viability was determined using SRB assay. Nitric oxide (NO) production was analyzed by Griess method and intracellular reactive oxygen species (ROS) by fluorescence analysis. In addition, cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) expression, MAPK activation and IkappaBalpha (IKBα) degradation, were determined by Western blot. ResultsAfter BFAC characterization, (+)-catechin was revealed as its major constituent. Both BFAC and (+)-catechin, exerted significant anti-oxidant and anti-inflammatory effects inhibiting LPS-induced intracellular ROS and NO production in peritoneal macrophages. Additionally, the extract but also its major component reduced pro-inflammatory proteins expression probably through c-Jun N-terminal kinase and p38 MAPK signaling pathways. ConclusionThese data suggest that the beneficial effects of BFAC might be mediated, at least in part, by the presence of (+)-catechin. Conclusively our findings confirm the potential of A. cochliacarpos as a new therapeutic strategy for the management of inflammatory and oxidative stress-related diseases.

Single-walled carbon nanotubes and graphene oxides induce autophagosome accumulation and lysosome impairment in primarily cultured murine peritoneal macrophages

The wide application of carbon nanomaterials in various fields urges in-depth understanding of the toxic effects and underlying mechanisms of these materials on biological systems. Cell autophagy was recently recognized as an important lysosome-based pathway of cell death, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles, but the underlying mechanisms are still uncertain due to the fact that autophagosome accumulation can result from autophagy induction and/or autophagy blockade. In this study, we first evaluated the toxicity of acid-functionalized single-walled carbon nanotubes and graphene oxides, and found that both carbon nanomaterials induced adverse effects in murine peritoneal macrophages, and GOs were more potent than AF-SWCNTs. Both carbon nanomaterials induced autophagosome accumulation and the conversion of LC3-I to LC3-II. However, degradation of the autophagic substrate p62 protein was also inhibited by both nanomaterials. Further analyses on lysosomes revealed that both carbon nanomaterials accumulated in macrophage lysosomes, leading to lysosome membrane destabilization, which indicates reduced autophagic degradation. The effects of AF-SWCNTs and GOs on cell autophagy revealed by this study may shed light on the potential toxic mechanism and suggest caution on their utilization.

Extracts of Crinum latifolium inhibit the cell viability of mouse lymph oma cell line EL4 and induce activation of anti-tumour activity of macrophages in vitro

Ethnopharmacological relevanceCrinum latifolium L. (CL) leaf extracts have been traditionally used in Vietnam and are now used all over the world for the treatment of prostate cancer. However, the precise cellular mechanisms of the action of CL extracts remain unclear. Aim of the studyTo examine the effects of CL samples on the anti-tumour activity of peritoneal murine macrophages. Materials and methodsThe properties of three extracts (aqueous, flavonoid, alkaloid), one fraction (alkaloid), and one pure compound (6-hydroxycrinamidine) obtained from CL, were studied (i) for redox capacities (DPPH and bleaching beta-carotene assays), (ii) on murine peritoneal macrophages (MTT assay) and on lymphoma EL4-luc2 cells (luciferine assay) for cytotoxicity, (iii) on macrophage polarization (production of ROS and gene expression by PCR), and (iv) on the tumoricidal functions of murine peritoneal macrophages (lymphoma cytotoxicity by co-culture with syngeneic macrophages). ResultsThe total flavonoid extract with a high antioxidant activity (IC50=107.36mg/L, DPPH assay) showed an inhibitory action on cancer cells. Alkaloid extracts inhibited the proliferation of lymphoma cells either by directly acting on tumour cells or by activating of the tumoricidal functions of syngeneic macrophages. The aqueous extract induced mRNA expression of tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin 6 (IL-6) indicating differentiation of macrophages into pro-inflammatory M1 polarized macrophages. The total flavonoid, alkaloid extracts and an alkaloid fraction induced the expression of the formyl peptide receptor (FPR) on the surface of the polarized macrophages that could lead to the activation of macrophages towards the M1 phenotype. Aqueous and flavonoid extracts enhanced NADPH quinine oxido-reductase 1 (NQO1) mRNA expression in polarized macrophages which could play an important role in cancer chemoprevention. All the samples studied were non-toxic to normal living cells and the pure alkaloid tested, 6-hydroxycrinamidine, was not active in any of the models investigated. ConclusionsOur results indicate that CL extracts and alkaloid fraction (but not pure 6-hydroxycrinamidine) inhibit the proliferation of lymphoma cells in multiple pathways. Our results are in accordance with traditional usage and encourage further studies and in vivo assays.

Pulchellamin G, an amino acid-sesquiterpene lactone, from Saussurea pulchella suppresses lipopolysaccharide-induced inflammatory responses via heme oxygenase-1 expression in murine peritoneal macrophages

Saussurea pulchella (Asteraceae) is widely distributed in Korea and has been used in Korean folk medicine for the treatment of inflammation, hypertension, hepatitis, and arthritis. Pulchellamin G is an amino acid-sesquiterpene lactone conjugate isolated from S. pulchella. In the present study, we focused on the anti-inflammatory effect of pulchellamin G, which acts by inducing the expression of heme oxygenase (HO)-1. HO-1 plays important roles in cytoprotection since it has antioxidant, anti-inflammatory, antiproliferative, and antiapoptotic properties. Pulchellamin G inhibited the mRNA and protein expression of inducible nitric oxide synthase (iNOS), iNOS-derived nitric oxide (NO), and cyclooxygenase (COX)-2 and COX-derived prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. The compound also reduced tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production and suppressed the phosphorylation and degradation of IκB-α and nuclear translocation of p65 in murine peritoneal macrophages in response to LPS stimulus. The inhibitory effects of pulchellamin G on nuclear factor kappa B (NF-κB) translocation was impaired by co-treatment of the cells with HO activity inhibitor tin protoporphyrin (SnPP). By using SnPP, we verified that the inhibitory effects of pulchellamin G on the pro-inflammatory mediators NO, PGE2, TNF-α, and IL-1β are associated with induction of HO-1 expression. Our data suggest that pulchellamin G might have potent therapeutic effects and it should be considered in the development of treatments for various inflammatory diseases.

Asymmetric dimethylarginine regulates the lipopolysaccharide-induced nitric oxide production in macrophages by suppressing the activation of NF-kappaB and iNOS expression

Two major effector systems are frequently implicated in the immune and endothelial cell alternations associated with inflammation. They include the enhanced production of reactive oxygen species and diminished bioavailability of nitric oxide (NO). Importantly, these processes can be regulated by endogenously produced methylarginines, inhibitors for NO derived from macrophages and endothelial cells. Therefore, the aim of this study was to show the potential pharmacological intervention of methylarginines (NG-methyl-L-arginine, L-NMMA; NG, NG′-dimethyl-L-arginine—symmetric dimethylarginine, SDMA; and NG, NG-dimethyl-L-arginine—asymmetric dimethylarginine, ADMA) in activation of murine peritoneal (RAW 264.7) and alveolar (MHS) macrophages with lipopolysaccharide from Gram-negative bacteria (LPS). The data presented in this study clearly declare that L-NMMA (1–50μM) and ADMA (10–50μM) significantly inhibited the LPS-induced NO production from macrophages in a concentration-dependent manner. It was demonstrated, for the first time, that the ADMA- and L-NMMA-induced down regulation of NO production was accompanied by reduced expression of mRNA and protein for inducible NO synthase as well as decreased activation of nuclear factor-κB. Importantly, we found a negative correlation between the ADMA-dependent reduction of NO production and ADMA-increased superoxide formation, which indicates that ADMA can negatively affect the balance in LPS-induced macrophage-derived production of reactive mediators. The only effect of SDMA was observed for LPS-triggered superoxide production, which was significantly decreased in its highest concentration (50μM). In summary, L-NMMA and ADMA can mediate their effects on macrophage activation via regulation of intracellular signaling pathways, which can affect critical functions in activated macrophages.

Salt-dependent chemotaxis of macrophages (P5122)

Abstract Recent studies support the hypothesis that macrophages are not only essential for efficient immune responses, but are also regulators of local tissue electrolyte composition and hence arterial hypertension. Blockade of this macrophage-dependent regulatory axis resulted in skin electrolyte accumulation and blood pressure increase. Based on the observation that macrophages accumulate in skin tissue of rats fed under high salt diet conditions, the question arose whether macrophages actively migrate to sites of high sodium storage. We therefore investigated the migratory behavior of RAW264.7 macrophages, murine bone marrow-derived macrophages and murine peritoneal macrophages toward different NaCl gradients, showing that NaCl may act as a chemotactic stimulus per se. We also assessed the reorganization of the actin cytoskeleton, a potential NaCl-induced chemokine CCL2 production and the role of the osmoprotective transcription factor TonEBP in salt-dependent chemotaxis. Although the underlying mechanism remains unidentified, we propose that salt-dependent chemotaxis might be an important functional feature of macrophages within the context of excess salt clearance in vivo.

송엽 에탄올 추출물의 아토피 저해 활성

The aim of this study was to examine inhibitory effects of pine needle ethanol extracts (PNEE) on atopic dermatitis (AD). To determine inflammatory activity PNEE was added to LPS-induced murine peritoneal macrophages for an in-vitro test. In addition, anti-AD test was carried out by spreading PNEE on the dorsal skin of 2,4-dinitrochlorobenzene (DNCB)- induced BALB/c mice. It was confirmed that the nitric oxide (NO) secretion was suppressed when <TEX>$1{\sim}50{\mu}g/mL$</TEX> of PNEE were added to LPS-induced murine peritoneal macrophages. Moreover, levels of TNF-<TEX>${\alpha}$</TEX>, IL-6, and IL-<TEX>$1{\beta}$</TEX>, were decreased. For the anti-AD test, PNEE alleviated symptoms of the erythema in DNCB-induced mice. Furthermore, the IFN-<TEX>${\gamma}$</TEX> secretion of the group treated with PNEE was increased in splenocytes from DNCB-induced mice compared to the positive control, while IL-4 secretion diminished. Through these results, we can conclude that PNEE can inhibit AD by modulating the IFN-<TEX>${\gamma}$</TEX>, IL-4 cytokines production and inhibiting inflammation.

Production of LPS-induced inflammatory mediators in murine peritoneal macrophages: neocuproine as a broad inhibitor and ATP7A as a selective regulator

Copper chelation regulates the production of inflammatory mediators in vivo during vascular inflammation and atherogenesis. Little is known about how the copper egress pump ATP7A regulates the production of these mediators. In this study, we isolated ATP7A deficient macrophages (MΦ) from the peritoneal cavity of blotchy mice and identified the lipopolysaccharide (LPS)-induced inflammatory mediators that were altered by ATP7A deficiency. These results were compared with the effect of neocuproine (a copper chelator) treatment on both ATP7A deficient and control MΦ. Seven of the 24 inflammatory mediators examined in this study had significant changes in expression in the ATP7A deficient MΦ compared to controls; 16 of these mediators were significantly reduced in MΦ treated with neocuproine compared to controls. Both neocuproine treatment and ATP7A deficiency reduced IFN-γ, MCP-1, MCP-3, and VEGF-A levels. Interestingly, the production of KC/GRO was upregulated by ATP7A deficiency but downregulated by neocuproine treatment. Neocuproine, but not ATP7A deficiency, reduced the production of FGF-9, IL-1α, IL-12p70, IL-2, IL-3, IL-4, IL-6, MIP-1β, MIP-2, RANTES, and TNFα. ATP7A deficiency but not neocuproine treatment reduced IP-10 and MCP-5 levels. In addition, both ATP7A deficiency and neocuproine treatment had no effect on GM-CSF, IL-10, IL-11, IL-7, OSM, and SCF. Together, these findings provide evidence that MΦ ATP7A selectively regulates LPS-induced inflammatory mediators, in part, via modulation of cellular copper availability, whereas neocuproine generally inhibits the production of inflammatory mediators. These results also imply that although copper chelation and ATP7A downregulation may result in different copper concentrations, gradients, and/or distribution in the cells, they may not lead to opposite biological effects on inflammatory mediator production.

Abstract 464: Tumor derived vacuolar ATPase subunit promotes M2 type polarization of macrophages.

Abstract Tumors and tumor associated macrophages (TAMs) express a unique surface vacuolar ATPase (V-ATPase) on the plasma membrane. In tumor cells, a 20 kd peptide is cleaved from a2 isoform of V-ATPase and is referred to as a2NTD; it is associated with in vitro induction of pro-tumorigenic properties in monocytes and IL-β secretion. In this study, we evaluated the immuno-modulatory properties of a2NTD in inducing murine peritoneal macrophage polarization and activation of inflammasome components both ex vivo and in vivo. Peritoneal macrophages from BALB/c mice were stimulated with recombinant a2NTD and the mRNA and protein expression of M1 (CD11c, TNF-α), M2 (Mannose receptor-1, Arginase-1, IL-10) markers, IL-1β and NLRP3 (inflammasome marker) were analyzed using quantitative real time PCR and flow cytometry. Further, mice were injected with a2NTD i.p. and peritoneal cells were analyzed for M1/M2 specific markers. The results of both in vivo and ex vivo analysis showed an elevated population of M2 subtype in a2NTD-treated mice compared to control (P&amp;lt;0.05). a2NTD upregulated the expression of IL-1β and NLRP3 in macrophages. These data demonstrated that a2NTD modulates macrophage polarization towards the M2 subtype functionally similar to tumor associated macrophages (TAMs). Since, tumor progression requires sterile inflammation and maturation of resident macrophages into TAMs; our data provide evidence that the surface expression of V-ATPase in tumor cells has a role in inducing the unique inflammatory environment of tumors by modulating macrophage phenotype and inducing IL-1β production through the action of a2NTD. The study also provides indication for the involvement of a2NTD in NLRP3 mediated inflammasome activation. Citation Format: Gajendra K. Katara, Mukesh K. Jaiswal, Alice Gilmam-Sacks, Kenneth D. Beaman. Tumor derived vacuolar ATPase subunit promotes M2 type polarization of macrophages. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 464. doi:10.1158/1538-7445.AM2013-464