Cell Line J774 Research Articles

Characterization and cell material interactions of PEGylated PNIPAAM nanoparticles

The aim of this study was to investigate the haemocompatibility of poly(N-isopropylacrylamide)-co-poly(ethylene glycol), PNIPAAM-PEG based nanoparticles and the influence of poly(ethylene glycol), PEG on the interactions of nanoparticles with cells. To achieve this purpose, thermosensitive PNIPAAM-PEG nanoparticles were synthesized by free radical dispersion polymerization method. Optimized nanosystems had particle sizes less than 200 nm, low polydispersity and LCST of 40–41 °C. The nanoparticles also showed nearly 83% encapsulation efficiency for doxorubicin HCl with temperature dependent release. Presence of PEG resulted in reduced protein adsorption by more than 50% in comparison to non-PEG containing nanoparticles. Protein adsorption was noted to be dependent on PEG chain length and was the least with M n = 4000. The particles up to a concentration of 2 mg/ml did not show any toxicity on J774 and L929 cell lines. No interactions were observed when NIPAAM-PEG nanoparticles were incubated with blood cells viz. RBCs, neutrophils, platelets and the coagulation system suggesting their haemocompatibility.

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.

Bone resorptive activity of osteoclast-like cells generated in vitro by PEG-induced macrophage fusion

Normal bone remodeling is maintained by a balance between osteoclast and osteoblast activity, whereas defects in osteoclast activity affecting such balance result in metabolic bone disease. Macrophage-macrophage fusion leading to multinucleated osteoclasts being formed is still not well understood. Here we present PEG-induced fusion of macrophages from both U937/A and J774 cell lines and the induced differentiation and activation of osteoclast-like cells according to the expression of osteoclast markers such as tartrate resistant acid phosphatase (TRAP) and bone resorptive activity. PEG-induced macrophage fusion, during the non-confluent stage, significantly increased the osteoclastogenic activity of macrophages from cell lines compared to that of spontaneous cell fusion in the absence of PEG (polyethylene glycol). The results shown in this work provide evidence that cell fusion per se induces osteoclast-like activity. PEG-fused macrophage differential response to pretreatment with osteoclastogenic factors was also examined in terms of its ability to form TRAP positive multinucleated cells (TPMNC) and its resorptive activity on bovine cortical bone slices. Our work has also led to a relatively simple method regarding those previously reported involving cell co-cultures. Multinucleated osteoclast-like cells obtained by PEG-induced fusion of macrophages from cell lines could represent a suitable system for conducting biochemical studies related to basic macrophage fusion mechanisms, bone-resorption activity and the experimental search for bone disease therapeutic alternatives.

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.

Investigation of the Immuno-Modulatory Effects of Aflatoxin B1, B2 and G1

ISEE-0309 Background and Objective: Mycotoxins are a diverse group of compounds that are produced by fungi of the Penicillium, Fusarium, and Aspergillus species. They occur in food and feed products which have fungal contamination. Mycotoxins can have severe detrimental effects on both human and animal health, including liver damage, cancer, and immunosuppression. It has been postulated that chronic aflatoxin exposure affects disease progression in individuals infected with HIV. The aim of this project was to investigate immunomodulatory effects of aflatoxins, in an effort to gain a better understanding of the mechanisms through which these compounds exert their immunosuppressive effects. Methods: Murine macrophages (cell line J774) were cultured in the presence of AFB1, AFB2, AFG1, singly or in combination, for up to 72 hours. Cells were treated with lipopolysaccharide (LPS) for 24 hours to induce a cytokine response. The effect of these aflatoxins on the secretion of the pro-inflammatory cytokines interleukin 1β (IL-1β), IL-12p40, IL-6 and Tumour Necrosis Factor-α (TNF-α), and the anti-inflammatory cytokine IL-10, were determined by Enzyme Linked Immuno-Sorbent Assay (ELISA). Results: There was a varied effect on the cytokine profile depending on the type of aflatoxin. All three aflatoxins tested had significant, dose-specific effect on levels of IL-12p40 after 24 hours treatment. AFG1 induced a significant increase in IL-6 secretion at low doses. Treatment with aflatoxin for 72 hours resulted in a statistically significant, dose-dependent increase in the secretion of the pro-inflammatory cytokine IL-6, and a decrease in the secretion of the anti-inflammatory cytokine IL-10. Conclusions: The data clearly show that the aflatoxins have immunomodulatory effects on macrophages. The effect of the aflatoxins on IL-6 and IL-10 highlight the pro-inflammatory nature of these compounds. These results help explain the effects of aflatoxin exposure such as cancer, liver damage, and vaccine failure.

Anti-nitric oxide production activity of isothiocyanates correlates with their polar surface area rather than their lipophilicity

There is increasing demand for novel anti-inflammatory drugs with different mechanisms of action. We synthesized a series of isothiocyanates 2b– h based on 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) found in the pungent spice Wasabia japonica. Inhibitory activities against in-vitro growth of tumor cells and production of nitric oxide (NO) using the mouse macrophage-like cell line J774.1 were noted. All isothiocyanates were optimized by Hartree-Fock/3-21G model, and the log P values and the polar surface area (PSA) values were calculated. Substitution of the methylsulfinyl group (CH 3S( O)–R) in 6-MITC with a formyl (CHO–R), a methylsulfanyl (CH 2S–R) or a methyl (CH 3–R) group reduced the activities of the parent isothiocyanate. Substitution with a formyl group resulted in lower lipophilicity (log P value) whereas substitution with a methylsulfanyl or methyl group resulted in a lower PSA value. The inhibitory activity of isothiocyanates showed better correlation with their PSA values rather than their partition coefficient (log P) values. Isothiocyanates with higher PSA values and some degree of log P value may have potent biological activity.

Cytotoxic activity ofEnterobacter cloacaehuman isolates

We examined 55 Enterobacter cloacae isolates from clinical specimens for the production of cytotonic and cytotoxic toxins and the presence of the type III secretion system (TTSS). Twelve isolates (22%) revealed cytotoxic activity that caused destruction of Vero cells, whereas 28 (51%) strains induced lysis of the murine macrophage J774 cell line. TTSS genes were present in 27% of the isolates. The results indicated that these bacteria may destroy phagocytes and epithelial cells, which may lead to spread within the host.

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.

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.

Inhibition of iNOS and COX-2 in human whole blood ex vivo and monocyte-macrophage J774 cells by a new group of aminothiopyrimidone derivatives

We tested a series of 11 new aminothiopyrimidones on the activity of inducible nitric oxide synthase (iNOS) and prostaglandin G/H synthase-1 and 2 (COX-1 and COX-2) in the whole human blood and monocyte-macrophage J774 cell line. To induce COX-2 and iNOS, blood samples and J774 cells were stimulated with bacterial lipopolysaccharide (LPS) in the absence or presence of the test compounds. After incubation, the plasma and the supernatants of culture media were collected for the measurement of TxB 2 and PGE 2 by a specific enzyme-immunoassay and determination of nitrite by a colorimetric assay. Several phenylthieno derivatives of substituted pyrimidone inhibited formation of both COX-2 and iNOS derived products with one of the compounds (compound 11, N-[2-[(2,4-dinitrophenyl)thio]-4-oxo-6-phenylthieno[2,3- d]pyrimidin-3(4 H)-y]methanesulfonamide) showing a complete inhibition of LPS-stimulated formation of NO and PGE 2.

Regulators of G‐protein signalling are modulated by bacterial lipopeptides and lipopolysaccharide

Regulators of G-protein signalling accelerate the GTPase activity of G(alpha) subunits, driving G proteins in their inactive GDP-bound form. This property defines them as GTPase activating proteins. Here the effect of different Toll-like receptor agonists on RGS1 and RGS2 expression in murine bone marrow-derived macrophages and J774 cells was analysed. After stimulation with TLR2/1 or TLR2/6 lipopeptide ligands and the TLR4/MD2 ligand lipopolysaccharide, microarray analyses show only modulation of RGS1 and RGS2 among all the regulators of G-protein signalling tested. Real-time PCR confirmed modulation of RGS1 and RGS2. In contrast to RGS2, which was always downregulated, RGS1 mRNA was upregulated during the first 30 min after stimulation, followed by downregulation. Similar results were also found in the murine macrophage cell line J774. The ligand for intracellular TLR9 modulates RGS1 and RGS2 in a similar manner. However, the TLR3 ligand poly(I:C) permanently upregulates RGS1 and RGS2 expression indicating a different modulation by the MyD88- and TRIF-signalling pathway. This was confirmed using MyD88(-/-) and TRIF(-/-) bone marrow-derived macrophages. Modulation of RGS1 and RGS2 by Toll-like receptor ligands plays an important role during inflammatory and immunological reactions after bacterial and viral infection.

Porphyromonas gingivalis with either Tannerella forsythia or Treponema denticola induces synergistic IL-6 production by murine macrophage-like J774.1 cells

Background Chronic periodontitis is caused by mixed bacterial infection. Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are frequently detected in deep periodontal pockets. We demonstrate that these bacteria induce proinflammatory cytokine production by the mouse macrophage-like cell line J774.1. Materials and methods J774.1 cells were incubated with and without bacteria for 24 h in 96-well flat-bottomed plates. The culture supernatants were analyzed by enzyme-linked immunosorbent assay for secreted mouse interleukin (IL)-6, monocyte chemoattractant protein-1, IL-23, IL-1β and tumor necrosis factor-α. The cytokine concentrations were determined using a standard curve prepared for each assay. Results Mixed infection with P. gingivalis and either T. forsythia or T. denticola at 10 5 CFU/ml acted synergistically to increase IL-6 production, but not monocyte chemoattractant protein-1, IL-23, IL-1β or tumor necrosis factor-α production. Gingipain inhibitors KYT-1 and KYT-36 inhibited IL-6 production by J774.1 cells incubated with 10 5 CFU/ml of mixed bacteria. Conclusion These results suggest that P. gingivalis with either T. forsythia or T. denticola directly induces synergistic IL-6 protein production and that gingipains play a role in this synergistic effect.

The Effect of Interferon-γ and Lipopolysaccharide on the Growth of Francisella tularensis LVS in Murine Macrophage-like Cell Line J774

Background:Francisella tularensis, a causative agent of human tularemia, displaying the ability to proliferate inside the human cells. Aims: To evaluate the growth potential ofF. tularensisLVS strain in macrophage-like cell line J774 modulated by recombinant interferon γ andE. coliderived lipopolysaccharide. Results: Stimulation of J774 cells either by interferon-γ or lipopolysaccharide alone, or especially in combination before infectionF. tularensis, revealed protective effects. Higher concentrations of stimulating agents were needed to inhibit ongoingF. tularensisinfection. Conclusions: Stimulation of J774 cell line by combination of interferon-γ with lipopolysaccharide inhibits the intracellular growth ofF. tularensis.

Inhibition of leukotriene B 4 production in murine macrophages by lactic acid bacteria

Leukotriene B 4 (LTB 4) is a lipid mediator associated with innate immune function. LTB 4 is produced mainly by polymorphonuclear leukocytes and macrophages and plays important roles in host defense. However, LTB 4 is also associated with inflammation, and excessive production of LTB 4 can result in inflammation, allergic reactions, and carcinogenesis. Regulation of excessive LTB 4 production may therefore assist in controlling these conditions. In this study, we investigated the capacity of 7 strains of lactic acid bacteria to inhibit LTB 4 production by the murine macrophage cell line J774.1. All strains tested inhibited calcium ionophore (A23187)-stimulated LTB 4 production in macrophages, but to varying extents. Lactobacillus helveticus Bc-10 exhibited the highest level of inhibitory activity. The inhibitory activity of strain Bc-10 was sustained after heat-killing and was observed in the intracellular cell-free extract prepared from this strain. This is the first report that intact lactic acid bacteria and their isolated cellular components can directly inhibit LTB 4 production by macrophages, and provides a useful method to screen the inhibitory activity of lactic acid bacteria. This study also highlights the potential of the strain Bc-10 as a treatment option for the regulation of LTB 4 production in vivo.

Isotope tracing enhancement of chemiluminescence assays for nitric oxide research

Chemiluminescence assays are used widely for the detection of nitric oxide (NO)-derived species in biological fluids and tissues. Here, we demonstrate that these assays can be interfaced with mass-sensitive detectors for parallel determination of isotopic abundance. Results obtained with tri-iodide and ascorbic acid-based reductive assays indicate that mass spectrometric detection enables NO isotope-tracing experiments to be carried out to a limit of detectability of a few picomoles, a sensitivity similar to that of standard gas phase chemiluminescence methods. The advantage afforded by mass spectrometric detection is demonstrated using the murine macrophage cell line J774, which is shown here to reduce 15NO3- to 15NO2- under anoxic conditions. The particular combination of an analytical and cellular system described here may hold promise for future characterization of the enzymatic pathways contributing to mammalian nitrate reductase activity, without background interference from 14NO2- derived from other sources.

Surface Modified Ultrathin Polyelectrolyte Nanoreservoir for Delivery of Proteins: Evaluation in Terms of Controlled Release and Biocompatibility

AbstractThe proposed ultrathin polyelectrolyte nanoreservoir (UPN) was fabricated in two combinations by alternate layering of polyelectrolytes poly (allylamine hydrochloride) along with sodium poly (styrene sulfonate) (PAH/PSS)5 and sodium alginate (PAH/SA)5 using porous calcium carbonate as a template using layer-by-layer adsorption technique with the subsequent template removal at low pH. We studied the possibility whether remnant intact nanoreservoir could be suited for encapsulation as well as delivery vehicle for protein such as bovine serum albumin as a model. To tune biocompatibility with biological cells the assembled surface was modified using pluronic (F-68) by adsorption and possible hydrophobic interaction. The prepared system was characterized for surface morphology, size and size distribution, surface charge, layer-by-layer growth due to sequential adsorption and surface modification. The experimental data obtained by Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared Spectroscopy (FTIR) provide evidence for the stepwise surface modification of the films. Further the system was investigated for payload efficiency of proteins, in-vitro release profile, integrity of proteins, cell adhesion and viability against biological cells (murine macrophages cell line J774.1). In both formulations polyelectrolyte composition lead to smooth and spherical nanomatrix, with payload of approx. 72±6% of proteins. Both the systems exhibited biphasic release profile with initial burst release followed by controlled release with overall release of 43.63±4.8% and 44±5.76% in 48h for formulation prepared by combination of (PAH/PSS)5 and (PAH/SA)5 respectively. There was a marked reduction in cell adhesion and improvement in viability upon surface modification. In a nutshell, the proposed system could successfully be used for the delivery of proteins and moreover the system can be tailored to impart desired properties at any stage of layering especially in terms of drug release and to retain the integrity of proteins.

Survival of stress exposed Campylobacter jejuni in the murine macrophage J774 cell line

Although campylobacters are relatively fragile and sensitive to environmental stresses, Campylobacter jejuni has evolved mechanisms for survival in diverse environments, both inside and outside the host. Their survival properties and pathogenic potential were assessed after subjecting food and clinical C. jejuni isolates to different stress conditions. After exposure to starvation (5 h and 15 h of nutrient depletion), a temperature shock (3 min at 55 °C) or oxidative stress (5 h and 15 h of atmospheric oxygen) we studied the culturability, viability and capability of adhesion, internalization and survival within the in vitro cell culture model using J774 murine macrophages. Starvation severely impaired C. jejuni culturability, particularly after 15 h of nutrient depletion. The number of viable cells decreased by 30–40%. Starved bacterial cells also showed a lower capability of adhesion, internalization and survival within macrophages. Despite the reduced culturability and viability of the heat treated cells, C. jejuni efficiently adhered to, and entered murine macrophages. However, the number of heat treated cells started to decrease more quickly than non-stressed cells. Within 24 h post infection all the cells were killed. The bacterial mechanisms involved in inactivating toxic oxygen products may enhance bacterial persistence through increased binding, entry and survival of both oxidatively stressed C. jejuni isolates inside the macrophages. Oxygen exposure increased the internalization and intracellular survival, although the cells cannot remain viable for extended periods within murine macrophages. However, any prolongation of survival in macrophages may increase the probability of transmission of bacteria in the host organism and have further implications in the pathogenesis of campylobacteriosis. This indicates that environmental stress conditions may be involved.