Murine Macrophage-like J774 Research Articles

Inhibition of the uric acid efflux transporter ABCG2 enhances stimulating effect of soluble uric acid on IL-1β production in murine macrophage-like J774.1 cells.

Soluble uric acid (UA) absorbed by cells through UA transporters (UATs) accumulates intracellularly, activates the NLRP3 inflammasome and thereby increases IL-1β secretion. ABCG2 transporter excludes intracellular UA. However, it remains unknown whether ABCG2 inhibition leads to intracellular accumulation of UA and increases IL-1β production. In this study, we examined whether genetic and pharmacological inhibition of ABCG2 could increase IL-1β production in mouse macrophage-like J774.1 cells especially under hyperuricemic conditions. We determined mRNA and protein levels of pro-IL-1β, mature IL-1β, caspase-1 and several UATs in culture supernatants and lysates of J774.1 cells with or without soluble UA pretreatment. Knockdown experiments using an shRNA against ABCG2 and pharmacological experiments with an ABCG2 inhibitor were conducted. Extracellularly applied soluble UA increased protein levels of pro-IL-1β, mature IL-1β and caspase-1 in the culture supernatant from lipopolysaccharide (LPS)-primed and monosodium urate crystal (MSU)-stimulated J774.1 cells. J774.1 cells expressed UATs of ABCG2, GLUT9 and MRP4, and shRNA knockdown of ABCG2 increased protein levels of pro-IL-1β and mature IL-1β in the culture supernatant. Soluble UA increased mRNA and protein levels of ABCG2 in J774.1 cells without either LPS or MSU treatment. An ABCG2 inhibitor, febuxostat, but not a urate reabsorption inhibitor, dotinurad, enhanced IL-1β production in cells pretreated with soluble UA. In conclusion, genetic and pharmacological inhibition of ABCG2 enhanced IL-1β production especially under hyperuricemic conditions by increasing intracellularly accumulated soluble UA that activates the NLRP3 inflammasome and pro-IL-1β transcription in macrophage-like J774.1 cells.

Immunostimulatory activity of snake fruit peel extract on murine macrophage-like J774.1 cells

Snake fruit (Salacca edulis Reinw.) is a tropical fruit produced in Indonesia. Snake fruit peel is normally discarded as waste. In the present study, it was revealed that snake fruit peel has high bioactivities on stimulation of the immune system. Snake fruit peel extract (SFPE) was prepared by extracting snake fruit peel powder in water for 15h at 4°C. SFPE enhanced phagocytotic activity of murine macrophage-like J774.1 cells. Production of cytokine such as tumour necrosis factor (TNF)-α and interleukin (IL)-6 was also stimulated by SFPE. The gene expression levels for these cytokines were elevated. Immunoblot analysis revealed that SFPE enhanced not only nuclear factor (NF)-κB but also mitogen-activated protein kinases signalling cascades such as JNK and p38 in macrophage. Overall findings suggested that SFPE has a potential beneficial effect to promote our body health through the stimulation of macrophage.

CD13 mediates phagocytosis in human monocytic cells

The myelomonocytic marker aminopeptidase N/CD13 is a novel phagocytic receptor in monocytes and macrophages.

Jellyfish collagen stimulates maturation of mouse bone marrow-derived dendritic cells

Jellyfish has recently attracted much attention for various applications, including functional foods due to its abundance of collagen. Jellyfish collagen, extracted from Nemopilema nomurai, was found to stimulate murine macrophage-like J774.1 cells. However, few reports have determined the immunostimulatory effects of jellyfish collagen on the innate immune response. We herein demonstrate the effect of jellyfish collagen on mouse bone marrow-derived dendritic cells (BMDCs). Jellyfish collagen stimulated TNF-α, IL-6, IL-1β and IL-12 production by BMDCs as the result of the elevation of gene expression level of these cytokines. In addition, jellyfish collagen-treated BMDCs have more wrinkles and longer pseudopodia on the cell surface compared with the control cells. Jellyfish collagen also stimulated cell-surface MHC-II expression level. Furthermore, jellyfish collagen downregulated phagocytosis capacity of BMDCs. Thus, our findings suggest that JC has the potential to activate DCs and thereby contribute to health promotion.

Gd-containing conjugated polymer nanoparticles: bimodal nanoparticles for fluorescence and MRI imaging.

Aqueous bifunctional semiconductor polymer nanoparticles (SPNs), approximately 30 nm in diameter (as measured from electron microscopy), were synthesised using hydrophobic conjugated polymers, amphiphilic phospholipids and a gadolinium-containing lipid. Their fluorescence quantum yields and extinction coefficients were determined, and their MRI T₁-weighted relaxation times in water were measured. The bimodal nanoparticles were readily taken up by HeLa and murine macrophage-like J774 cells as demonstrated by confocal laser scanning microscopy, and were found to be MRI-active, generating a linear relationship between T₁-weighted relaxation rates and gadolinium concentrations The synthesis is relatively simple, and can easily result in milligrams of materials, although we fully expect scale-up to the gram level to be easily realised.

Lysosomal iron in pulmonary alveolar proteinosis: a case report

Pulmonary alveolar proteinosis is characterised by accumulation of surfactant-like material in the distal air spaces. Since lysosomes play a crucial role for degradation of large biomolecules taken up from the cell's environment, it was hypothesised that oxidant-induced lysosomal disruption and ensuing cell death might play a role in disease development. In the present study, alveolar macrophages, harvested by whole-lung lavage from a patient diagnosed with pulmonary alveolar proteinosis, are shown to contain large amounts of undigested material within lysosomes, and the same organelle exhibits increased amounts of haemosiderin-bound iron. Compared with murine macrophage-like J774 cells (iron exposed or not), the status of human macrophages was pro-oxidative, i.e. macrophages exhibited a low level of the antioxidant glutathione and large amounts of iron available for Fenton-type chemistry. As a consequence, macrophageal lysosomes were particularly fragile when exposed to physiological concentrations of hydrogen peroxide (generated by glucose oxidase in culture medium). Such lysosomal disruption resulted in extensive cell death by both necrosis and apoptosis independent of caspase-3 activation. Considering the potential role of iron-catalysed oxidant-induced lysosomal rupture and ensuing cell killing for pulmonary alveolar proteinosis pathology and disease progression, whole-lung lavage might be considered early in those cases in which cytochemical staining reveals great numbers of haemosiderin-laden alveolar macrophages.

The influence of the immunostimulation by bacterial cell components derived from altered large intestinal microbiota on probiotic anti-inflammatory benefits

Using murine macrophage-like J774.1 cells and fecal precipitates prepared from the feces of elderly volunteers whose acute inflammation had been inhibited by LKM512 yogurt consumption, we investigated the likelihood that immunostimulation by altered intestinal bacterial cell components contribute to the anti-inflammatory benefits of this yogurt. Tumor necrosis factor-alpha production due to stimulation by fecal precipitates obtained during LKM512 yogurt consumption tended to be higher than due to stimulation by precipitates obtained from preconsumption (P=0.0827), although acute phase response was suppressed by LKM512 yogurt consumption. We suggest that the anti-inflammatory benefits of LKM512 yogurt on elderly volunteers are independent of direct immunostimulation by the bacterial cell components derived from altered intestinal microbiota.

Anti-Inflammatory Metabolite Production in the Gut from the Consumption of Probiotic Yogurt ContainingBifidobacterium animalissubsp.lactisLKM512

There is little evidence for a relationship between probiotic metabolites and host cytokine production. We investigated in the present study the possibility that anti-inflammatory metabolites can be produced in the gut by LKM512 yogurt consumption by using murine macrophage-like J774.1 cells and extracts prepared from the feces of elderly volunteers. These volunteers' acute inflammation had been inhibited by LKM512 yogurt consumption in a previous test. The tumor necrosis factor (TNF)-alpha production elicited in J774.1 cells stimulated by lipopolysaccharide (LPS) and in the fecal extracts obtained during the period of LKM512 yogurt consumption was significantly decreased (p<0.05) than the pre-consumption baseline level. These findings and previous data enable us to conclude that intestinal bacterial metabolites produced by LKM512 yogurt consumption contributed to suppressing the inflammatory cytokine produced by macrophages and that one of the anti-inflammatory metabolites in the fecal extracts was likely to have been a polyamine.

Characterization of a flocculation-like phenotype in Cryptococcus neoformans and its effects on pathogenesis

We investigated the phenomenon of cell-cell aggregation (flocculation) in a serotype D strain of Cryptococcus neoformans (ATCC 24067, isolate RC-2). Cell aggregation into clumps of 5-40 cells (clump+ cells) occurred during the early log phase and disappeared in the beginning of the stationary phase (clump- cells). The cell aggregation phenomenon was medium dependent. Clump+ cells could be dispersed by either vortexing or proteinase K digestion. Most importantly, the transient change in cellular phenotype changed several important host-pathogen interactions. Adherence of clump+ cells to murine macrophage-like cells J774.16 was significantly (P < 0.001) enhanced compared with adherence of clump- cells. Furthermore, complement-mediated phagocytosis efficacy of dispersed clump+ cells was significantly higher (P < 0.001) compared with clump- cells. Similar findings were documented with an in vivo phagocytosis assay. Infection of mice with a low inoculum (10(4)) of clump+ cells resulted in lower fungal burden when compared with mice infected with clump- cells. Accordingly, mice infected with clump+ cells survived significantly longer than mice infected with clump- cells. These results indicate that the cellular phenotype undergoes significant changes that result in a transient flocculation-like phenotype. We hypothesize that this cell-cell aggregation is the result of changes in protein content in the polysaccharide capsule. We conclude from our data that the change in cellular phenotype has a dramatic effect on cell adherence, and on complement-mediated phagocytosis, both of which can affect the pathogenesis of the disease in the host. Our results underscore the complexity of studies that investigate host pathogen interactions and may explain differences and inconsistencies observed in in vitro and in vivo assays.

Rhodococcus equi can survive a phagolysosomal environment in macrophages by suppressing acidification of the phagolysosome

Rhodococcus equi is one of the most important causes of pneumonia in foals and has emerged as a significant opportunistic pathogen of immunosuppressed hosts such as human immunodeficiency virus-infected patients. Virulent R. equi harbouring an 85 kb plasmid, but not the avirulent form lacking the plasmid, has the ability to survive in macrophages. However, the survival mechanism is not known. In the present study, morphological interactions were observed between virulent or plasmid-cured avirulent R. equi and phagolysosomes in murine macrophage-like J774.1 cells by immunocytological methods. The J774.1 cells phagocytosed virulent and avirulent bacteria to a similar extent, and both bacteria replicated in single membrane vacuoles at similar rates up to 6 h after infection. Thereafter, the virulent bacteria continued to grow, whereas the avirulent bacteria stopped growing. When the infected cells were stained with phagosomal and lysosomal markers and observed with a confocal fluorescence microscope, the majority of phagosomes containing these bacteria were fused with lysosomes. Neither R. equi organism has the ability to hinder phagosome-lysosome fusion. The acidity in phagolysosomes containing R. equi was examined by staining with LysoTracker Red DND-99, an acidotropic probe. The phagolysosomes containing virulent organisms were not acidic as compared with avirulent organisms. Over 90% of the phagolysosomes containing avirulent R. equi were stained with LysoTracker 6 h after infection, whereas less than 50% of those containing virulent R. equi were stained. Furthermore, when the supernatant obtained from a virulent R. equi culture was added to the cell cultures, the acidity of acidic compartments in macrophages was reduced. The authors conclude that some substance(s) produced by virulent R. equi suppress acidification in phagolysosomes, and help R. equi survival and replication in the bactericidal environment.

Human cytochrome c enters murine J774 cells and causes G 1 and G 2/M cell cycle arrest and induction of apoptosis

Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c 551 from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c 551, the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G 1 to S phase, as well as at the G 2/M phase at higher concentrations. Unlike P. aeruginosa cytochrome c 551 which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G 2/M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 °C, suggesting energy requirement in the entry process.

Involvement of Reactive Oxygen Intermediate in the Enhanced Expression of Virulence‐Associated Genes of Listeria monocytogenes inside Activated Macrophages

Listeriolysin O encoded by 1,587 bp hly is the essential virulence factor of Listeria monocytogenes that replicates in the cytosolic space after escaping from phagosome of macrophages. By using murine macrophage-like J774.1 cells with or without activation by IFN-gamma plus LPS, the expression of both hly and its positive regulator prfA was monitored by means of RT-PCR. In activated J774.1 cells, the level of hly expression was enhanced although the multiplication of bacteria was significantly suppressed. The elevated expression of hly inside activated macrophage was abolished by addition of SOD and catalase, suggesting that reactive oxygen intermediates contribute to the upregulation of prfA and hly transcriptions. Moreover, we found that exposure of L. monocytogenes to H2O2 dramatically enhanced the expression of both prfA and hly mRNAs. Spontaneous ONOO- generator, SIN-1, also promoted the transcription to a certain level. These results suggested that oxygen radicals generated in activated macrophages provide a positive signal for up-regulation of virulence genes in L. monocytogenes.

Bisphosphonate Induced Growth Inhibition of Breast Cancer Cells is Augmented by p38 Inhibition

Bisphosphonates (BPs) inhibit osteoclast-mediated bone resorption. They also protect from cancer-induced osteolysis and inhibit breast cancer growth in vitro. Some breast cancer cell lines, however, are relatively resistant to the growth inhibitory effects of BPs. We studied the mechanism of BP resistance in human MDA-MB-231 breast cancer cells. We show that both pyrophosphate-resembling (p-) and nitrogen-containing (n-) BPs induce activation of p38 mitogen activated protein (MAP) kinase pathway in MDA-MB-231 cells in vitro. MDA-MB-231 cells stably expressing a dominant negative form of the p38 MAP kinase (p38/AF) exhibited a dramatic increase in growth inhibition in response to BPs in vitro, compared to control cells. SB203580, a specific inhibitor of the p38 MAP kinase, also augmented BP-induced growth inhibition of parental MDA-MB-231 cells. Similar results were obtained also in murine macrophage-like J774 cells in vitro. Finally, no BP-induced phosphorylation of p38, or augmentation of BP-induced growth inhibition by SB203580 were detected in MCF-7 or HCC38 breast cancer cells, which are more sensitive than MDA-MB-231 cells especially to n-BP induced growth inhibition. In conclusion, these results suggest that BPs activate the p38 pathway in MDA-MB-231 and J774 cells. This activation may be associated with increased survival or proliferation because inhibition of p38 augments BP-induced growth inhibition. These findings may apply to the development of novel approaches for the treatment of breast cancer.

Caffeic acid phenethyl ester (CAPE) analogues: potent nitric oxide inhibitors from the Netherlands propolis.

The MeOH and water extracts of the Netherlands propolis were tested for their inhibitory activity toward nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage-like J774.1 cells. Both of the extract possessed significant NO inhibitory activity with IC(50) values of 23.8 and 51.5 microg/ml, respectively. Then 13 phenolic compounds obtained from the MeOH extract showing stronger NO inhibition were examined on their NO inhibitory activities. Caffeic acid phenethyl ester (CAPE) analogues, i.e., benzyl caffeate, CAPE and cinnamyl caffeate, possessed most potent NO inhibitory activities with IC(50) values of 13.8, 7.64 and 9.53 microM, respectively, which were two- to four-fold stronger than the positive control N(G)-monomethyl-L-arginine (L-NMMA; IC(50), 32.9 microM). Further study on the synthetic analogues of CAPE revealed that both of 3-phenylpropyl caffeate (18; IC(50), 7.34 microM) and 4-phenylbutyl caffeate (19; IC(50), 6.77 microM) possessed stronger NO inhibitory activity than CAPE (10) and that elongation of alkyl side chain of alcoholic parts of caffeic acid esters enhance the NO inhibitory activity. In addition, it was found that CAPE analogues having longer carbon chain (>C(5)) in alcoholic part showed toxic effects toward J774.1 cells. This NO inhibitory effect may directly correlate with antiinflammatory properties of the Netherlands propolis.

Structural and biological characterisation of a novel tetra-acyl lipid A from Escherichia coli F515 lipopolysaccharide acting as endotoxin antagonist in human monocytes

We here report on the structural analysis of a novel tetra-acyl lipid A (LA (tetra) ) isolated from Escherichia coli deep rough (Re)-mutant strain F515. In addition to the biologically active hexa-acyl E. coli-type lipid A (compound 506), this incompletely acylated lipid A was found to be also present in the native LPS. Its structure was studied without further derivatisation by chemical analysis, matrix-assisted laser desorption/ionization mass spectrometry, and one- and two-dimensional (1)H- and (13)C-NMR spectroscopy. It was found to be structurally distinct from the tetra-acyl lipid A biosynthetic precursor Ia (compound 406) in lacking the primary (R)-3-hydroxytetradecanoic acid 14:0(3-OH) in position 3' ester-linked to the 'non-reducing' glucosamine (GlcN II). The hydroxyl group at the (R)-3-hydroxytetradecanoic acid attached to position 2' of GlcN II was found to be substituted by dodecanoic acid (12:0), thus forming a dodecanoyloxytetradecanoyl residue 14:0[3-O(12:0)]. The acylation pattern at the 'reducing' GlcN I was identical to that of compound 406 in having two primary (R)-3-hydroxy tetradecanoic acid residues [14:0(3-OH)] attached to positions 3 (ester-linked) and 2 (amide-linked), respectively. In human mononuclear cells (hMNC) the new LA (tetra) antagonized LPS-induced release of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in a dose-dependent manner with identical antagonistic potency as compared with compound 406. Also like compound 406, it was found to be an agonist in murine macrophage-like J774.1 cells.

Quinolactacins A, B and C: novel quinolone compounds from Penicillium sp. EPF-6. I. Taxonomy, production, isolation and biological properties.

Quinolactacins A (1), B (2) and C (3), novel quinolone antibiotics have been found from the cultured broth of a fungal strain isolated from the larvae of the mulberry pyralid Margaronia pyloalis Welker). The fungal strain, EPF-6 was identified as Penicillium sp. from its morphological characteristics. Quinolactacins were obtained from the culture medium by solvent extraction and chromatographic purification. Compound 1 showed inhibitory activity against tumor necrosis factor (TNF) production induced by murine peritoneal macrophages and macrophage-like J774.1 cells stimulated with lipopolysaccharide (LPS).

Enhancement of nitric oxide synthesis by macrophages represents an additional mechanism of action for amphotericin B.

Amphotericin B (AmB) enhanced nitrite synthesis by murine macrophage-like J774.16 cells in a dose-dependent fashion. This effect was retained in the presence of Cryptococcus neoformans capsular polysaccharide, a known virulence factor. AmB and anticapsular antibody increased nitrite synergistically. In all cases, AmB required gamma interferon; C. neoformans cells were unable to elicit nitrite, with or without AmB.

Macrophage Activation in Response to S-Form Lipopolysaccharides (LPS) Separated by Centrifugal Partition Chromatography from Wild-Type LPS: Effects of the O-Polysaccharide Portion of LPS

The S-form lipopolysaccharide (LPS) was effectively separated from a native preparation of smooth-type Salmonella abortus equi LPS by means of the centrifugal partition chromatography (CPC). To clarify the mechanisms by which LPS activates macrophages, CPC-separated S-form LPS was assessed for its ability to induce the secretion of tumor necrosis factor-α (TNF-α) by murine macrophage-like J774.1 cells in comparison with other fractions of LPS which lacks most of O-polysaccharides. LPS dose-response and time-kinetics studies showed that serum factor(s) regulated especially the onset of TNF-α secretion in stimulation with S-form LPS. These results strongly suggest that the native (unfractionated) LPS activates macrophages in both O-polysaccharide/serum-dependent and -independent pathways.

Immune complexes increase nitric oxide production by interferon-gamma- stimulated murine macrophage-like J774.16 cells.

Murine macrophage-like J774.16 cells were tested for changes in nitric oxide production upon incubation with immune complexes. Cryptococcus neoformans capsular polysaccharide and polysaccharide-specific monoclonal antibodies were added to J774.16 cells in the presence and absence of recombinant murine interferon-gamma (IFN-gamma). The effect of immune complexes on nitrite synthesis was both concentration dependent and isotype dependent. In the presence of IFN-gamma, immune complexes of IgG1, IgG2, IgG2b, or IgG3 isotype increased nitrite levels, whereas complexes of IgM isotype did not. Immune complexes did not alter nitrite production by unstimulated macrophages. Antibody alone, antigen alone, and antigen with irrelevant IgG1 antibody did not augment nitrite formation, either in the presence or absence of IFN-gamma, indicating a requirement for Fc gamma R cross-linking. These results suggest that IgG isotypes may offer additional protection against pathogens by enhancing macrophage nitric oxide production.

Antibodies to Cryptococcus neoformans glucuronoxylomannan enhance antifungal activity of murine macrophages

Monoclonal antibodies (MAbs) to the capsular polysaccharide of the pathogenic fungus Cryptococcus neoformans can prolong survival and decrease organ fungal burden in experimental murine cryptococcosis. To investigate the mechanism of antibody-mediated protection, the interaction of C. neoformans and murine macrophage-like J774.16 cells was studied in the presence and absence of MAbs differing in isotype. Immunoglobulin G2a (IgG2a) and IgG2b isotype switch variants were isolated from an IgM hybridoma to complete the IgG subclass set. IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA MAbs were studied for their ability to promote phagocytosis and reduce the number of CFU in C. neoformans and J774.16 cell cocultures. The MAbs in this set had similar if not identical fine specificities and were derived from a single B cell. All isotypes promoted phagocytosis; however, the IgG subclasses were more effective opsonins than IgM or IgA. All isotypes enhanced J774.16 anti-C. neoformans activity in vitro, as measured by a reduction in the number of CFU. The IgG1 MAbs were consistently more active in promoting opsonization and reducing the number of CFU. Addition of IgG1 MAb to C. neoformans and J774.16 cocultures resulted in rapid reduction in the number of CFU, which is consistent with fungal killing. Electron microscopy revealed that MAb-opsonized C. neoformans cells were internalized and appeared damaged. Administration of IgM, IgG1, IgG2a, and IgG2b isotype switch variant MAbs revealed that the IgG2a and IgG2b subclasses were the most and least effective isotypes, respectively, in prolonging survival in an intraperitoneal murine infection model. The results indicate that murine antibody subclasses differ in their ability to enhance macrophage anti-C. neoformans activity and suggest that antibody enhancement of macrophage function is a mechanism by which antibodies modify infection in vivo.