Macrophage Cell Line J774 Research Articles

In vitropro- and anti-inflammatory responses to viableCandida albicansyeasts by a murine macrophage cell line

The balance between pro- and anti-inflammatory cytokine production is a crucial aspect of infections caused by Candida albicans. We therefore investigated the effect of yeast concentration on the pro- and anti-inflammatory cytokine response. Production of tumor necrosis factor-alpha (TNF-α) by the murine macrophage cell line J774 decreased significantly as the live yeast concentration increased. In parallel, a dose-dependent production of interleukin-10 (IL-10) was observed with live C. albicans. Paraformaldehyde-treated yeasts were unable to stimulate or modulate this response. Yeast culture supernatants induced high levels of TNF-α production when added to the macrophage cells, but did not down-modulate the macrophage response. In contrast, supernatants from yeast-macrophage co-cultures stimulated less TNF-α production and induced a down-modulation of the macrophage response, showing that modulation depended on macrophage- or yeast-derived components secreted when the yeast concentration was high. Phospholipomannan (PLM), but not farnesol, stimulated TNF-α production, but neither PLM nor farnesol down-modulated cytokine production in response to yeasts. Yeast culture supernatants or supernatants from yeast-macrophage co-cultures did not induce IL-10 production. These results suggest that the final macrophage cytokine response is determined by the interplay between live yeasts and macrophages, and that the direction of the response is dependent on the yeast concentration.

PEGylation and preliminary biocompatibility evaluation of magnetite–silica nanocomposites obtained by high energy ball milling

High energy ball milling (HEBM) has been used for the first time to prepare PEGylated magnetite–silica (Fe 3O 4–SiO 2) nanocomposites intended to be used for biological purposes. Surface amine groups were introduced by a silanization reaction involving 3-aminopropyl triethoxysilane (APTS) followed by PEGylation to yield long-term stable and stealth nanocomposites of 200 nm in diameter. The efficient coverage by PEG chains was shown by isothermal titration calorimetry (ITC) where PEGylated nanocomposites did not interact with BSA compared to non-PEGylated counterparts which led to a significant change in enthalpy. By cell viability (MTT) assays and cell morphology investigations, it was evidenced that PEGylated Fe 3O 4–SiO 2 nanocomposites did not provide any appreciable cytotoxicity on J774 macrophage and MCF-7 breast cancer cell lines. Furthermore, noticeable internalization was evidenced by J774 cells with PEGylated Fe 3O 4–SiO 2 nanocomposites in contrast to MCF-7 cells, in good agreement with the respective tendency of each cell line for endocytosis.

Changes in biphasic electrode impedance with protein adsorption and cell growth

This study was undertaken to assess the contribution of protein adsorption and cell growth to increases in electrode impedance that occur immediately following implantation of cochlear implant electrodes and other neural stimulation devices. An in vitro model of the electrode–tissue interface was used. Radiolabelled albumin in phosphate buffered saline was added to planar gold electrodes and electrode impedance measured using a charge-balanced biphasic current pulse. The polarization impedance component increased with protein adsorption, while no change to access resistance was observed. The maximum level of protein adsorbed was measured at 0.5 µg cm−2, indicating a tightly packed monolayer of albumin molecules on the gold electrode and resin substrate. Three cell types were grown over the electrodes, macrophage cell line J774, dissociated fibroblasts and epithelial cell line MDCK, all of which created a significant increase in electrode impedance. As cell cover over electrodes increased, there was a corresponding increase in the initial rise in voltage, suggesting that cell cover mainly contributes to the access resistance of the electrodes. Only a small increase in the polarization component of impedance was seen with cell cover.

Role of MyD88 and Toll-like receptors 2 and 4 in the sensing of Parachlamydia acanthamoebae.

Parachlamydia acanthamoebae is a Chlamydia-related organism whose pathogenic role in pneumonia is supported by serological and molecular clinical studies and an experimental mouse model of lung infection. Toll-like receptors (TLRs) play a seminal role in sensing microbial products and initiating innate immune responses. The aim of this study was to investigate the roles of MyD88, TLR2, and TLR4 in the interaction of Parachlamydia with macrophages. Here, we showed that Parachlamydia entered bone-marrow derived macrophages (BMDMs) in a TLR-independent manner but did not multiply intracellularly. Interestingly, compared to live bacteria, heat-inactivated Parachlamydia induced the production of substantial amounts of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and IL-12p40 by BMDMs and of TNF and IL-6 by peritoneal macrophages as well as RAW 264.7 and J774 macrophage cell lines. Cytokine production by BMDMs, which was partially inhibited upon trypsin treatment of Parachlamydia, was dependent on MyD88, TLR4, and, to a lesser extent, TLR2. Finally, MyD88(-/-), TLR4(-/-), and TLR2(-/-) mice were as resistant as wild-type mice to lung infection following the intratracheal instillation of Parachlamydia. Thus, in contrast to Chlamydia pneumoniae, Parachlamydia acanthamoebae weakly stimulates macrophages, potentially compensating for its low replication capacity in macrophages by escaping the innate immune surveillance.

BAS/BSCR55 Diverse bacteria promote macrophage foam cell formation: potential role of Toll-like receptor signalling pathways

We and others have shown that the molecular signatures of diverse bacterial species accumulate in human atheromatous lesions. Here, we aimed to determine the effect of non-viable bacteria on foam...

Monocyte chemoattractant protein-1/CC chemokine ligand 2 enhances apoptotic cell removal by macrophages through Rac1 activation

Apoptotic cell removal (efferocytosis) is an essential process in the regulation of inflammation and tissue repair. We have shown that monocyte chemoattractant protein-1/CC chemokine ligand 2 (MCP-1/CCL2) enhances efferocytosis by alveolar macrophages in murine bacterial pneumonia. However, the mechanism by which MCP-1 exerts this effect remains to be determined. Here we explored that hypothesis that MCP-1 enhances efferocytosis through a Rac1/phosphatidylinositol 3-kinase (PI3-kinase)-dependent mechanism. We assessed phagocytosis of apoptotic cells by MCP-1 treated murine macrophages in vitro and in vivo. Rac activity in macrophages was measured using a Rac pull down assay and an ELISA based assay (GLISA). The downstream Rac1 activation pathway was studied using a specific Rac1 inhibitor and PI3-kinase inhibitor in in vitro assays. MCP-1 enhanced efferocytosis of apoptotic cells by murine alveolar macrophages (AMs), peritoneal macrophages (PMs), the J774 macrophage cell line (J774s) in vitro, and murine AMs in vivo. Rac1 activation was demonstrated in these cell lines. The effect of MCP-1 on efferocytosis was completely negated by the Rac1 inhibitor and PI3-kinase inhibitor. We demonstrated that MCP-1 enhances efferocytosis in a Rac1–PI3 kinase-dependent manner. Therefore, MCP-1–Rac1–PI3K interaction plays a critical role in resolution of acute lung inflammation.

A Novel Biocompatible Poly (3-hydroxy-co-4-hydroxybutyrate) Blend as a Potential Biomaterial for Tissue Engineering

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) P(3HB-co-4HB) biopolymer was subjected to a depyrogenation treatment followed by blending with vitamin E (P(3HB-co-4HB)/Vit E blend), collagen (Col blend) or both (P(3HB-co-4HB)/Vit E/Col hybrid blend) to improve its biological performance. The cytocompatibility and pro-inflammatory response to the biopolymer after the treatment and blending were evaluated by cellular responses in vitro. Cell attachment and spreading of murine NIH 3T3 fibroblasts seeded on the films were found to be the highest on the hybrid P(3HB-co-4HB)/Vit E/Col blend film. The P(3HB-co-4HB) blends showed increased cell proliferation and negligible cytotoxicity. The pro-inflammatory cytokines, IL6 and TNF α expression in the murine macrophage cell line J774 A-1 monocyte/macrophage cells cultured on the biopolymer films were assessed by immunoblotting and RT-PCR analyses which revealed that the combined blending strategy was the most effective in reducing the pro-inflammatory activity. The depyrogenated P(3HB-co-4HB) blends significantly extend the range of biomaterials suitable for tissue engineering.

In-vitro study of cellular viability and nitric oxide production by J774 macrophages stimulated by interferon gamma with ceramic, polycarbonate, and polyoxymethylene brackets

Ceramic brackets are chemically inert in the oral cavity, whereas polycarbonate and polyoxymethylene brackets can degrade and release bisphenol-A and formaldehyde, respectively. More reliable tests are needed to assess the potential toxicity of these materials. In addition to traditional cytotoxicity tests, the study of nitric oxide (NO) cellular production stimulated by a specific material has been shown to be a reliable tool for evaluating cytotoxic potential. The purpose of this study was to assess, with esthetic brackets, cellular viability by 3,(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay (Sigma, St. Louis, Mo) in the macrophage cell line J774 stimulated with interferon gamma. Interferon gamma is a key cytokine in the activation of macrophages, plays an important role in immunologic processes, and also quantifies NO production by these macrophages. Well plates were seeded with 2 x 104 J774 cells per well, in a volume of 100 microL, resuspended in Roswell Park Memorial Institute Supplemented Medium 1640. The macrophage cell line J774 was stimulated with interferon gamma. Ceramic, polycarbonate, and polyoxymethylene brackets were added and kept in the culture for 24, 48, or 72 hours in 5% carbon dioxide at 37 degrees C; the control samples did not include brackets. At the end of each incubation period, the supernatant was collected for posterior NO quantification, and the cells were evaluated for cytotoxicity. Cellular viability in all groups was higher at 72 hours than at 24 hours. The final means in the bracket groups did not show significant differences compared with the control group. NO production was significantly greater in all groups at the final time than at the initial time. However, the brackets with the interferon gamma stimulation did not result in greater NO production than did the cells in the control group.

Surface functionalisation of PLGA nanoparticles for gene silencing

This work presents a method for decorating the surface of poly (lactide-co-glycolide) (PLGA) nanoparticles with polyethyleneimine (PEI) utilising a cetyl derivative to improve surface functionalisation and siRNA delivery. Sub-micron particles were produced by an emulsion-diffusion method using benzyl alcohol. We demonstrate by x-ray photoelectron spectroscopy (XPS), 2.6 times higher surface presentation of amines using the cetyl derivative compared to non-cetylated-PEI formulations (6.5 and 2.5% surface nitrogen, respectively). The modified particles were shown by spectroscopy, fluorescent microscopy and flow cytometry to bind and mediate siRNA delivery into the human osteosarcoma cell line U2OS and the murine macrophage cell line J774.1. Specific reduction in the anti-apoptotic oncogene BCL-w in U2OS cells was achieved with particles containing cetylated-PEI (53%) with no cellular toxicity. In addition, particles containing cetylated-PEI achieved 64% silencing of TNFα in J774.1 cells. This rapid method for surface modification of PLGA nanoparticles promotes its application for alternative cetylated functional derivatives as a strategy to control specific biological properties of nanoparticles.

Macrophage Nitrite Uptake and Release

Activated macrophages are known to produce large concentrations of nitrite from the synthesis of nitric oxide. With the recent discovery that nitrite is a biologically active substance, one question that gains more significance is how macrophages hold the nitrite they produce. In this respect, the question of how quickly macrophages internalize or externalize the anion becomes of relevance. Here we report on experiments that characterize the kinetics of nitrite uptake into, and release from the murine J774 macrophage cell line. Cells were exposed to 100 μM of 15N‐labeled nitrite and intracellular or extracellular concentrations measured as a function of time using a mass spectrometric method developed in our lab. Our results show that transport of the nitrite in and out of the cells is on the order of a minute of less, and that the ensuing intracellular and extracellular concentrations of the anion are similar. The results suggest that macrophage nitrite uptake and release is dominated by passive transport.

The G1 cell cycle arrest of macrophages infected with Aggregatibacter actinomycetemcomitans

Infection of murine macrophage cell line J774.1 with the periodontopathic bacterium Aggregatibacter actinomycetemcomitans induces apoptotic cell death. The infection induces cell cycle arrest in the G1 phase prior to the appearance of apoptotic cells. This study determined the involvement of various cell cycle-related signal molecules in A. actinomycetemcomitans-induced G1 cell cycle arrest. Cell cycle in J774.1 cells infected with A. actinomycetemcomitans was analyzed with a flow cytometer. Immunoblot analysis was also employed to determine the expression levels of intracellular signal molecules. Flow cytometric analysis revealed that the percentage of cells in the G1 phase increased to 77.2% at 12 h after A. actinomycetemcomitans infection. Additionally, according to immunoblot analysis, expression levels of hyperphosphorylated forms of retinoblastoma protein (ppRb) declined in J774.1 cells following A. actinomycetemcomitans infection, whereas hypophosphorylated Rb (pRb) expression levels were elevated slightly. Expression levels of cyclin D1 and D2 in the cells decreased gradually postinfection; CDK2, CDK4, CDK6 and cyclin E levels were not changed. Furthermore, postinfection, p21(CIP1/WAF1) expression increased at 6 h, followed by a subsequent decrease. These findings suggest that cyclin D1 and D2 and p21(CIP1/WAF1) participate in G1 cell cycle arrest in A. actinomycetemcomitans-infected J774.1 cells.

J774 macrophage-like cell line cytokine and chemokine patterns are modulated by Francisella tularensis LVS strain infection

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

In-vitro study of the cellular viability and nitric oxide production by J774 macrophages with ceramic, polycarbonate, and polyoxymethylene brackets

Studies show that ceramic brackets are chemically inert in the oral cavity, whereas polycarbonate and polyoxymethylene brackets can degrade, releasing bisphenol-A and formaldehyde, respectively. In addition to the traditional cytotoxicity tests, the study of nitric oxide cellular production stimulated by a specific material has been shown to be a reliable tool for evaluating its cytotoxic potential. We aimed to assess cellular viability by MTT (Sigma, St. Louis, Mo): 3,(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide assay in a murine macrophage cell line J774 with esthetic brackets and quantify nitric oxide production by these macrophages. Cell cultures were evaluated at 3 times: 24, 48, and 72 hours. Cellular viability in all groups was higher at 72 hours compared with 24 hours. This increase was significant in the control and ceramic brackets groups. Final means in the bracket groups showed no significant differences compared with the control group. Nitric oxide production was significantly greater in all groups at final time. There was no significant difference between the final means of the bracket groups and the control group, although polyoxymethylene brackets showed significantly greater means at 24 and 48 hours. Final means in the bracket groups showed no significant differences compared with the control group.

Cytotoxic Activity of Serratia marcescens Clinical Isolates

Twenty Serratia marcescens isolates from clinical specimens were examined for their cytotoxic activity on four cell lines (HEp-2, Vero, CHO, J774). Most of the isolates were found to be cytotoxic to CHO (70%), Vero (75%) and HEp-2 cells (90%). CHO cells were the most sensitive to cell-free supernatants, followed by HEp-2 and Vero cells. Two strains produced cytotonic toxins which caused elongation of CHO cells. Moreover, twelve isolates (60%) revealed cytotoxic potential to macrophage cell line J774. The results indicate that these bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host.

Inhibitory effects of fucose-related sugar compounds on oxidised low-density lipoprotein uptake in macrophage cell line J774.1

The interaction between oxidised low-density lipoprotein (LDL) and macrophages is known to be important in the development of arteriosclerosis. Macrophages take up oxidised LDL and then become foam cells, which contribute to the thickening of the blood vessel wall. In our previous paper, some flavonoids found in vegetables and fruits were shown to have a protective effect against arteriosclerosis. In this communication, to elucidate the effect of fucose-related compounds on oxidised LDL uptake in macrophages, the inhibitory activity of various fucose-related compounds on the 1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate-acetylated-LDL (DiI-ac-LDL) uptake reaction in mouse macrophage cell line J774.1 was measured. Fucoidan, 4-deoxy-fucose and fucose-galactose-N-acetyl-glucose significantly inhibited uptake of DiI-ac-LDL into macrophages. The IC50 of 4-deoxy-fucose was 108.4 µM, the highest value of the fucose-related compounds used in the experiments. The protective effects of the fucose-related compounds on atherosclerosis can be used as new health foods.

Yessotoxin inhibits phagocytic activity of macrophages

Yessotoxin (YTX) is a sulphated polyether compound produced by some species of dinoflagellate algae, that can be accumulated in bivalve mollusks and ingested by humans upon eating contaminated shellfish. Experiments in mice have demonstrated the lethal effect of YTX after intraperitoneal injection, whereas its oral administration has only limited acute toxicity, coupled with an alteration of plasma membrane protein turnover in the colon of the animals. In vitro studies have shown that this effect is due to the inhibition of endocytosis induced by the toxin. In this work, we investigated the effects of YTX on phagocytosis by using the J774 macrophage cell line. We found that macrophages exposed to 10 or 1 nM YTX display a reduced phagocytic activity against Candida albicans; moreover, phagosome maturation is also inhibited in these cells. Such results were confirmed with resident peritoneal macrophages from normal mice. The inhibition of both phagocytosis and phagosome maturation likely involves cytoskeletal alterations, since a striking rearrangement of the F-actin organization occurs in YTX-treated J774 macrophages. Surprisingly, YTX also enhances cytokine production (TNF-α, MIP-1α and MIP-2) by J774 macrophages. Overall, our results show that low doses of YTX significantly affect both effector and secretory functions of macrophages.

High Throughput Cell-Based Screening of Biodegradable Polyanhydride Libraries

A parallel screening method has been developed to rapidly evaluate discrete library substrates of biomaterials using cell-based assays. The biomaterials used in these studies were surface-erodible polyanhydrides based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH), and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) that have been previously studied as carriers for drugs, proteins, and vaccines. Linearly varying compositional libraries of 25 different polyanhydride random copolymers (based on CPH:SA and CPTEG:CPH) were designed, fabricated, and synthesized using discrete (organic solvent-resistant) multi-sample substrates created using a novel rapid prototyping method. The combinatorial libraries were characterized at high throughput using infrared microscopy and validated using 1H NMR and size exclusion chromatography. The discrete libraries were rapidly screened for biocompatibility using standard SP2/0 myeloma, CHO and L929 fibroblasts, and J774 macrophage cell lines. At a concentration of 2.8 mg/mL, there was no appreciable cytotoxic effect on any of the four cell lines evaluated by any of the CPH:SA or CPTEG:CPH compositions. Furthermore, the activation of J774 macrophages was evaluated by incubating the cells with the polyanhydride libraries and quantifying the secreted cytokines (IL-6, IL-10, IL-12, and TNFalpha). The results indicated that copolymer compositions containing at least 50% CPH induced elevated amounts of TNFalpha. In summary, the results indicated that the methodologies described herein are amenable to the high throughput analysis of synthesized biomaterials and will facilitate the rapid and rational design of materials for use in biomedical applications.

Surface-associated lipoprotein PpmA of Streptococcus pneumoniae is involved in colonization in a strain-specific manner

Streptococcus pneumoniae produces two surface-associated lipoproteins that share homology with two distinct families of peptidyl-prolyl isomerases (PPIases), the streptococcal lipoprotein rotamase A (SlrA) and the putative proteinase maturation protein A (PpmA). Previously, we have demonstrated that SlrA has PPIase activity, and that the enzyme plays a role in pneumococcal virulence. Here, we investigated the contribution of PpmA to pneumococcal pathogenesis. Pneumococcal mutants of D39 and TIGR4 lacking the gene encoding PpmA were less capable of persisting in the nasopharynx of mice, demonstrating the contribution of PpmA to pneumococcal colonization. This observation was partially confirmed in vitro, as the pneumococcal mutants NCTC10319DeltappmA and TIGR4DeltacpsDeltappmA, but not D39DeltacpsDeltappmA, were impaired in adherence to Detroit 562 pharyngeal cells. This suggests that the contribution of PpmA to pneumococcal colonization is not solely the result of its role in adherence to epithelial cells. Deficiency in PpmA did not result in reduced binding to various extracellular matrix and serum proteins. Similar to SlrA, we observed that PpmA was involved in immune evasion. Uptake of PpmA-deficient D39Deltacps and NCTC10319 by human polymorphonuclear leukocytes was significantly enhanced compared to the isogenic wild-types. In addition, ingestion of D39DeltappmA, but not that of either NCTC10319DeltappmA or TIGR4DeltappmA, by murine macrophage cell line J774 was also enhanced, whereas intracellular killing remained unaffected. We conclude that PpmA contributes to the early stages of infection, i.e. colonization. The contribution of PpmA to virulence can be explained by its strain-specific role in adherence to epithelial cells and contribution to the evasion of phagocytosis.

The Use Of Rap-PCR In Studying Mycobacterium tuberculosis Intracellular Gene During Macrophage Infection

Mycobacterium tuberculosis is the second leading cause of death from infectious agent. This study sought to detect M. tuberculosis genes, which were specifically expressed, or upregulated during intracellular infection of J774 murine macrophages; as such genes may be potential targets for novel drug action. J774 murine macrophage cell line was infected with M. tuberculosis (H37Rv strain) at 10:1 multiplicity of infection (MOI). RNA was differentially extracted from M. tuberculosis infecting J774 macrophage cell line. The control in this case was RNA from extracellular broth grown bacteria. Approximately 50 ng of RNA from intracellular derived bacteria and extracellular derived bacteria (control) were subjected to random arbitrarily primed PCR (RAP-PCR) using 50 primer combinations. Eleven differential RAP-PCR products were observed. All RAP-PCR products were cloned into pCR®2.1 and sequenced in order to determine the identity of the products. Four of the eleven products were derived from macrophage genes and another 4 products were derived from the M. tuberculosis rRNA genes (three 23S and one 16S rRNA). The 3 remaining RAP-PCR products were found to be mycobacterial genes other than ribosomal genes. The three products were genes encoding enzyme involving in a shikimate pathway, a putative carboxyphosphonoenolpyruvate phosphonomutase and a serine protease with homology to HtrA. Of the 3 mycobacterial genes other than ribosomal genes detected, none were specifically expressed during intracellular infection but competitive RT-PCR showed that aroF gene was upregulated two-fold in intracellular derived bacilli.

Synthesis and Comparison of Antimalarial Activity of Febrifugine Derivatives Including Halofuginone

Febrifugine and its derivatives including halofuginone which possess very high activity against malaria were prepared synthetically from easily available starting material, 3-hydroxy picoline, and using simple reaction conditions. Synthesis of 2-amino-5, 6-methylenedioxy benzoic acid, (which is an intermediate for the process) is described. The selectivity enhancement in nitration of 3, 4-methylenedioxybenzaldehyde towards 6-nitro isomer was done with the help of surfactant. The antimalarial activity of synthesized compounds was determined by using in vitro assays against chloroquine sensitive (D6), chloroquine resistant (W2) Plasmodium falciparum strains for susceptibility and two mammalian cell lines (neuronal cell line NG108 and macrophage cell line J774) for cytotoxicity. The IC(50)s of halofuginone was observed to be the best among the synthesized derivatives of febrifugine.