J774 Macrophage Cell Research Articles

Nitric oxide detection in cell culture exposed to LPS after Er:YAG laser irradiation

To evaluate in vitro the effect of calcium hydroxide [Ca(OH)(2)] and Er:YAG laser on bacterial endotoxin [also known as lipopolysaccharide (LPS)] as determined by nitric oxide (NO) detection in J774 murine macrophage cell line culture. Samples of LPS solution (50 microg mL(-1)), Ca(OH)(2) suspension (25 mg mL(-1)) and LPS suspension with Ca(OH)(2) were prepared. The studied groups were: I - LPS (control); II - LPS + Ca(OH)(2); III - LPS + Er:YAG laser (15 Hz 140 mJ); IV - LPS + Er:YAG laser (15 Hz 200 mJ); V - LPS + Er:YAG laser (15 Hz 250 mJ), VI - Pyrogen-free water; VII - Ca(OH)(2). Murine macrophage J774 cells were plated and 10 microL of the samples were added to each well. The supernatants were collected for NO detection by the Griess reaction. Data were analysed statistically by one-way anova and Tukey's test at 5% significance level. The mean and SE (in micromol L(-1)) values of NO release were: I - 10.48 +/- 0.58, II - 6.41 +/- 0.90, III - 10.2 +/- 0.60, IV - 8.35 +/- 0.40, V - 10.40 +/- 0.53, VI - 3.75 +/- 0.70, VII - 6.44 +/- 0.60; and the values for the same experiment repeated after 1 week were: I - 21.20 +/- 1.50, II - 9.10 +/- 0.60, III - 19.50 +/- 1.00, IV - 18.50 +/- 0.60, V - 21.30 +/- 0.90, VI - 2.00+/- 0.20, VII - 6.80 +/- 1.70. There was no significant difference (P > 0.05) between the control and the laser-treated groups (III, IV and V), or comparing groups II, VI and VII to each other (P > 0.05). Group I had significantly higher NO release than group II (P < 0.05). Groups II and VI had similar NO release (P > 0.05). Calcium hydroxide inactivated the bacterial endotoxin (LPS) whereas none of the Er:YAG laser parameter settings had the same effectiveness.

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.

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.

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.

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.

Discovery and SAR of cinnolines/quinolines as liver X receptor (LXR) agonists with binding selectivity for LXRβ

A series of cinnolines/quinolines was prepared and it was found that 4-phenyl-cinnoline/quinolines with either a 2′,3′ or 2′,5′-disubstituted benzyloxy moiety or the 1-Me-7-indole methoxy moiety on the meta position of the 4-phenyl ring showed good binding selectivity for LXRβ over LXRα. The LXRβ binding selective modulators displayed good activity for inducing ABCA1 gene expression in J774 macrophage cell line and poor efficacy in the LXRα Gal4 functional assay. 26, 37 and 41 were examined for their ability to induce SREBP-1c gene expression in Huh-7 liver cell line and they were weak partial agonists.

Scavenger Receptor A: A New Route for Adenovirus 5

Adenoviruses are common pathogens associated with respiratory diseases, gastrointestinal illnesses and/or conjunctivitis. Currently, this virus is used as a vector in gene therapy trials. The promise of viral gene therapy applications is substantially reduced because the virus is cleared by liver macrophages upon systemic administration. The mechanism underlying adenoviral tropism to and degradation in macrophages is poorly understood. We identified a new adenoviral receptor, the scavenger receptor A (SR-A), responsible for uptake of the virus in macrophages. CHO cells expressing SR-A showed increased viral transgene expression when compared with wild type cells. Preincubation of J774 macrophage cells with SR-A ligands decreased significantly adenoviral uptake. Electron-microscopy analysis of infected J774 cells showed activation of a viral degradation pathway. Infection of mice with adenovirus resulted in a substantial decrease of the virus in liver macrophages when SR-A was blocked. Our data provide a basis for understanding of the adenoviral uptake and degradation mechanism in macrophages in vitro and in vivo. Inhibition of adenoviral SR-A uptake can be utilized in gene therapy applications to increase its efficiency and efficacy.

Cadmium increases ferroportin-1 gene expression in J774 macrophage cells via the production of reactive oxygen species

Cadmium intoxication has been associated with the dysregulation of iron homeostasis. In the present study, we investigated the effect of cadmium on the expression of ferroportin 1 (FPN1), an important iron transporter protein that is involved in iron release from macrophages. When we incubated cadmium with J774 mouse macrophage cells, FPN1 mRNA levels were significantly increased in a dose- and time-dependent manner. Furthermore, the cadmium-induced FPN1 mRNA expression was associated with increased levels of FPN1 protein. On the other hand, cadmium-mediated FPN1 mRNA induction in J774 cells was completely blocked when cells were co-treated with a transcription inhibitor, acitomycin D. Also, cadmium directly stimulated the activity of the FPN1-promoter driven luciferase reporter, suggesting that the cadmium up-regulates FPN1 gene expression in a transcription-dependent manner. Finally, cadmium exposure to J774 macrophages increased intracellular reactive oxygen species (ROS) levels by ~ 2-fold, compared to untreated controls. When J774 cells were co-treated with antioxidant N-acetylcystein, the cadmium-induced FPN1 mRNA induction was significantly attenuated. In summary, the results of this study clearly demonstrated that cadmium increased FPN1 expression in macrophages through a mechanism that involves ROS production, and suggests another important interaction between iron and cadmium metabolism.

Novel N-aryl- and N-heteryl phenazine-1-carboxamides as potential agents for the treatment of infections sustained by drug-resistant and multidrug-resistant Mycobacterium tuberculosis

We investigated the in vitro activity of a new class of N-aryl and N-heteryl phenazine-1-carboxamide derivatives against Mycobacterium tuberculosis H37Rv and against drug-resistant ATCC M. tuberculosis strains. The activity against M. tuberculosis in J774 macrophage cells was also investigated. In most cases, minimum inhibitory concentrations (MICs) ranging between 0.19 mg/L and 0.79 mg/L were found, and comparable MIC values were obtained against 26 susceptible and 5 drug-resistant clinical isolates. Several derivatives were shown to be effective in inhibiting the growth both of susceptible and resistant strains at comparable concentrations. Results obtained indicate that these compounds could represent a promising class of agents useful for the treatment of M. tuberculosis infections caused by drug-resistant strains.

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.

Innovative serum-free medium for in vitro cultivation of promastigote forms of Leishmania species

We described a comparatively simple medium formula (CML) using common, available and reasonably priced ingredients that could be used in place of medium that requires calf serum enhancement for cultivation of Leishmania promastigote forms. This medium equivalently supported the growth of parasites at rates comparable with those obtained with serum supplemented RPMI-1640 medium. Leishmania promastigotes reproduced in CML exhibited moderate to high infectivity capacities when tested against J774 macrophage cell line. No significant difference was noted between Leishmania strains cultivated in the newly modified medium and those grown in RPMI-1640 medium in their cells infectivity and replication potentials. The use of new CML can easily take the place of other biphasic or liquid media because of its easy preparation and instantaneous use, reasonable price, availability of ingredients, and its long shelf life, which is 30-45 days. The fact that this medium is similar to other culture media as far as durability and quantity of produced parasites might give it an advantage over the other currently used media.

Caco-2 소장세포와 J774 대식세포에서 Hepcidin 호르몬이 철분 수송체 Ferroportin과 Divalent Metal Transporter 1의 유전자 발현에 미치는 영향

Dept. of Food & Nutrition, Kyung Hee University, Seoul 130-710, KoreaAbstractHepcidin is a peptide hormone produced by the liver, of which secretion is closely related to iron status in the body. However, little is known about the molecular mechanism(s) by which this peptide regulates body iron homeostasis. The purpose of this study was to determine the effects of hepcidin treatment within the physiological concentration range on the expressions of two different iron transporter proteins-ferroportin (FPN) and divalent metal transporter 1 (DMT1). Differentiated Caco-2 intestinal cells and macrophage J774 cells were treated with either synthetic hepcidin or hepcidin-rich fraction separated from human urine at the concentration of 10 nM and 100 nM for 24 hours. Results show that hepcidin treatment in differentiated Caco-2 cells or in J774 cells did not change the level of either FPN mRNA or DMT1 mRNA. On the other hand, hepcidin treatment at the dose of 100 nM significantly decreased the FPN protein levels and DMT1 protein levels in differentiated Caco-2 cells. Similarly, urinary hepcidin treatment (10 nM & 100 nM) also significantly decreased the levels of FPN and DMT1 proteins in J774 macrophage cells. These results showed that hepcidin might play an important role in the regulation of iron homeostasis by lowering the protein levels of iron transporter FPN and DMT1 both in enterocytes and in macrophage cells.Key words: iron homeostasis, ferroportin, divalent metal transporter 1, hepcidin

Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages

Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the liver, Kupffer cells are responsible for adenovirus degradation and contribute to the inflammatory response. As scavenger receptors present on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated the possible implication of these receptors in the clearance of the adenovirus vector. Polyinosinic acid [poly(I)], a scavenger receptor A ligand, was analysed for its capability to inhibit adenovirus uptake specifically in macrophages. In in vitro studies, the addition of poly(I) before virus infection resulted in a specific inhibition of adenovirus-induced gene expression in a J774 macrophage cell line and in primary Kupffer cells. In in vivo experiments, pre-administration of poly(I) caused a 10-fold transient increase in the number of adenovirus particles circulating in the blood. As a consequence, transgene expression levels measured in different tissues were enhanced (by 5- to 15-fold) compared with those in animals that did not receive poly(I). Finally, necrosis of Kupffer cells, which normally occurs as a consequence of systemic adenovirus administration, was prevented by the use of poly(I). No toxicity, as measured by liver-enzyme levels, was observed after poly(I) treatment. From our data, we conclude that poly(I) can prevent adenovirus sequestration by liver macrophages. These results imply that, by inhibiting adenovirus uptake by Kupffer cells, it is possible to reduce the dose of the viral vector to diminish the liver-toxicity effect and to improve the level of transgene expression in target tissues. In systemic gene-therapy applications, this will have great impact on the development of targeted adenoviral vectors.

Poly(vinyl alcohol) acetoacetate-based tissue adhesives are non-cytotoxic and non-inflammatory

Polymer-based tissue adhesives composed of poly(vinyl alcohol) acetoacetate (PVOH acac) and cross-linking amines were investigated for their effects on cell survival and inflammatory cell activation using in vitro mouse cell cultures. Cytotoxicity of tissue adhesives was evaluated by placing adhesives in direct contact with 3T3 fibroblast cells. Tissue adhesives formulated from PVOH acac and 3-aminopropyltrialkoxysilane (APS) were non-cytotoxic to fibroblasts; adhesives formulated from PVOH acac and aminated poly(vinyl alcohol) (PVOH amine) were also non-cytotoxic to fibroblasts. In contrast, a commercial adhesive composed of 2-octyl cyanoacrylate was highly cytotoxic to fibroblasts. The inflammatory potential of tissue adhesives was evaluated by exposing J774 macrophage cells to adhesives, and measuring TNF-alpha release from macrophages. PVOH acac-based tissue adhesives did not elicit inflammatory TNF-alpha release from macrophages. These results suggest that PVOH acac-based tissue adhesives are non-cytotoxic and non-inflammatory. Such tissue adhesives represent a promising technology for a variety of medical applications, including surgical wound closure and tissue engineering, and the results are also significant in the design of in vitro cell culture systems to study biomaterials.

Combined Enzymatic and Data Mining Approaches for Comprehensive Phosphoproteome Analyses: Application to Cell Signaling Events of Interferon-γ-Stimulated Macrophages

Protein phosphorylation is a central cell signaling event that underlies a broad spectrum of key physiological processes. Advances in affinity chromatography and mass spectrometry are now providing the ability to identify and quantitate thousands of phosphorylation sites simultaneously. Comprehensive phosphoproteome analyses present sizable analytical challenges in view of suppression effects of phosphopeptides and the variable quality of MS/MS spectra. This work presents an integrated enzymatic and data mining approach enabling the comprehensive detection of native and putative phosphopeptides following alkaline phosphatase digestion of titanium dioxide (TiO2)-enriched cell extracts. The correlation of retention times of more than 750 phospho- and dephosphopeptide pairs from J774 macrophage cell extracts indicated that removal of the phosphate groups can impart a gain or a loss in hydrophobicity that is partly explained by the formation of a salt bridge with proximal amino groups. Dephosphorylation also led to an average 2-fold increase in MS sensitivity that facilitated peptide sequencing. More importantly, alkaline phosphatase digestion enhanced the overall population of putative phosphopeptides from TiO2-enriched cell extracts providing a unique approach to profile multiphosphorylated cognates that would have remained otherwise undetected. The application of this approach is demonstrated for differential phosphoproteome analyses of mouse macrophages exposed to interferon-gamma for 5 min. TiO2 enrichment enabled the identification of 1143 phosphopeptides from 432 different proteins of which 125 phosphopeptides showed a 2-fold change upon interferon-gamma exposure. The use of alkaline phosphatase nearly doubled the number of putative phosphopeptides assignments leading to the observation of key interferon-gamma signaling events involved in vesicle trafficking, production of reactive oxygen species, and mRNA translation.

Dysferlin Deficiency Enhances Monocyte Phagocytosis: A Model for the Inflammatory Onset of Limb-Girdle Muscular Dystrophy 2B

Dysferlin deficiency causes limb-girdle muscular dystrophy type 2B (LGMD2B; proximal weakness) and Miyoshi myopathy (distal weakness). Muscle inflammation is often present in dysferlin deficiency, and patients are frequently misdiagnosed as having polymyositis. Because monocytes normally express dysferlin, we hypothesized that monocyte/macrophage dysfunction in dysferlin-deficient patients might contribute to disease onset and progression. We therefore examined phagocytic activity, in the presence and absence of cytokines, in freshly isolated peripheral blood monocytes from LGMD2B patients and in the SJL dysferlin-deficient mouse model. Dysferlin-deficient monocytes showed increased phagocytic activity compared with control cells. siRNA-mediated inhibition of dysferlin expression in the J774 macrophage cell line resulted in significantly enhanced phagocytosis, both at baseline and in response to tumor necrosis factor-alpha. Immunohistochemical analysis revealed positive staining for several mononuclear cell activation markers in LGMD2B human muscle and SJL mouse muscle. SJL muscle showed strong up-regulation of endocytic proteins CIMPR, clathrin, and adaptin-alpha, and LGMD2B muscle exhibited decreased expression of decay accelerating factor, which was not dysferlin-specific. We further showed that expression levels of small Rho family GTPases RhoA, Rac1, and Cdc 42 were increased in dysferlin-deficient murine immune cells compared with control cells. Therefore, we hypothesize that mild myofiber damage in dysferlin-deficient muscle stimulates an inflammatory cascade that may initiate, exacerbate, and possibly perpetuate the underlying myofiber-specific dystrophic process.

Effects of various metal ions on the gene expression of iron exporter ferroportin-1 in J774 macrophages

Macrophages play a key role in iron metabolism by recycling iron through erythrophagocytosis. Ferroportin-1 (FPN1) is a transporter protein that is known to mediate iron export from macrophages. Since divalent metals often interact with iron metabolism, we examined if divalent metals could regulate the expression of FPN1 in macrophages. J774 macrophage cells were treated with copper, manganese, zinc, or cobalt at 10, 50, or 100 µM for 16 to 24 h. Then, FPN1 mRNA and protein levels were determined by quantitative real-time PCR and Western blot analyses, respectively. In addition, effects of divalent metals on FPN1 promoter activity were examined by luciferase reporter assays. Results showed that copper significantly increased FPN1 mRNA levels in a dose-dependent manner. The copper-induced expression of FPN1 mRNA was associated with a corresponding increase in FPN1 protein levels. Also, copper directly stimulated the activity of FPN1 promoter-driven reporter construct. In contrast, manganese and zinc had no effect on the FPN1 gene expression in J774 cells. Interestingly, cobalt treatment in J774 cells decreased FPN1 protein levels without affecting FPN1 mRNA levels. In conclusion, our study results demonstrate that divalent metals differentially regulate FPN1 expression in macrophages and indicate a potential interaction of divalent metals with the FPN1-mediated iron export in macrophages.

Biological characterization of Sorona polymer from corn-derived 1,3-propanediol

The Sorona family of polymers, based on corn-derived 1,3-propanediol, have recently been developed as novel bio-based materials for use in plastics, films, and fibers. In the present study, Sorona polymers were investigated for their effects on cell survival and inflammatory cell activation using in vitro mouse cell cultures. Cytotoxicity of Sorona polymers was evaluated by placing material samples in direct contact with 3T3 fibroblast cells. Both Sorona plastic and Sorona films were non-cytotoxic to fibroblasts. The inflammatory potential of Sorona samples was evaluated by exposing J774 macrophage cells to material samples, and measuring TNF-alpha release from macrophages. Sorona plastic and Sorona films did not elicit inflammatory TNF-alpha release from macrophages. These results indicate that Sorona polymers are non-cytotoxic and non-inflammatory. While the 1,3-propanediol component of Sorona 3GT is manufactured in a bacterial fermentation process, the absence of an inflammatory response to Sorona film and Sorona plastic is highly encouraging. The results are significant for the design of materials that utilize bio-based polymers.

Hepcidin inhibits apical iron uptake in intestinal cells

Hepcidin (Hepc) is considered a key mediator in iron trafficking. Although the mechanism of Hepc action in macrophages is fairly well established, much less is known about its action in intestinal cells, one of the main targets of Hepc. The current study investigated the effects of physiologically generated Hepc on iron transport in Caco-2 cell monolayers and rat duodenal segments compared with the effects on the J774 macrophage cell line. Addition of Hepc to Caco-2 cells or rat duodenal segments strongly inhibited apical (55)Fe uptake without apparent effects on the transfer of (55)Fe from the cells to the basolateral medium. Concurrently, the levels of divalent metal transporter 1 (DMT1) mRNA and protein in Caco-2 cells decreased while the mRNA and protein levels of the iron export transporter ferroportin did not change. Plasma membrane localization of ferroportin was studied by selective biotinylation of apical and basolateral membrane domains; Hepc induced rapid internalization of ferroportin in J774 cells but not in Caco-2 cells These results indicate that the effect of Hepc is cell dependent: in macrophages it inhibits iron export by inducing ferroportin degradation, whereas in enterocytes it inhibits apical iron uptake by inhibiting DMT1 transcription. Our results highlight the crucial role of Hepc in the control of intestinal iron absorption.

Abstract 912: Spleen Tyrosine Kinase Regulates TLR4 dependent Macropinocytosis And Lipid Accumulation In Macrophages

Toll-like receptors play a key role in the signaling pathways of innate immune response and have been recently shown to contribute to vascular inflammation and atherosclerosis. Oxidation of LDL is considered a leading mechanism of atherogenesis, and in our previous studies, we demonstrated that minimally modified LDL (mmLDL) induced TLR4-dependent chemokine secretion as well as robust actin polymerization and spreading of macrophages. We noticed that these mmLDL-induced cytoskeletal rearrangements led to extensive vacuolization, characteristic of macropinocytosis. In this study, we examined the mechanisms of TLR4-induced actin polymerization and macropinocytosis. It has been reported that spleen tyrosine kinase (Syk) regulates actin cytoskeleton and that it may interact with TLR4. We found in the immunoprecipitation experiments that mmLDL induced Syk association with TLR4 as well as Syk phosphorylation. Next we developed a J774 macrophage cell line, which stably expressed Syk shRNA and had the Syk expression reduced by 70% or more. Using this Syk-deficient macrophage cell line and specific Syk inhibitors, we demonstrated that Syk is necessary for mmLDL-induced activation of Ras and phosphorylation of Raf-MEK1-ERK1/2. The ERK1/2 activation resulted in phosphorylation of paxillin and an increase in the formation of N-WASP/Arp2 complex, which initiates actin polymerization. Taken together, these results suggest that Syk is a critical kinase in the mmLDL macrophage activation and that the mmLDL-induced cytoskeleton signaling. In addition, we demonstrated that mmLDL induced macrophage uptake of native LDL and of fluorescent 10 KDa dextran, and that this effect was Syk-dependent. These data suggest a novel pathway for the mmLDL-induced macropinocytosis in macrophages, which may constitute a mechanism for macrophage lipid accumulation and foam cell formation, a characteristic step in the development of atherosclerosis.