Induction of apoptosis in macrophage cell line,J774, by the cell-free supernatant from Pseudomonas aeruginosa

Pseudomonas aeruginosa is able to secrete many virulence factors that are cytotoxic towards eukaryotic cells. To investigate the effect of the bacterium on macrophages, we obtained cell-free supernatants from P. aeruginosa (Pa) IID1117 (elastase-positive and protease-positive) and Pa IID1130 (elastase-positive and protease-negative). After 6 hr of incubation with the cell-free supernatant from the Pa IID1117 strain, the viability of J774 macrophages was shown to be significantly reduced (47.5+/-11%), but not Pa IID1130 (96.4+/-1.6%) at a concentration of 10% (v/v) compared to control J774 macrophages without any supernatant (97.2+/-1.7%) by the detection of trypan blue dye exclusion. The death of cells was further demonstrated to be due to apoptosis characterized by chromatin condensation and apoptotic bodies by Hoechst 33258 staining, DNA fragmentation by agarose gel electrophoresis and terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL). An activated subunit was found to be released from procaspase-3 in cell lysate. But in the presence of protease inhibitor, the apoptosis was completely blocked. The findings indicate that the Pa IID1117 strain is capable of inducing apoptosis in J774 macrophages. The apoptosis induced by the cell-free supernatant from Pa IID1117 strain is suggested to be dependent on protease, but not elastase.     

Metabolism of cholesteryl ester lipid droplets in a J774 macrophage foam cell model

J774 macrophages rapidly incorporated [3H]cholesteryl oleate droplets by a non-saturable phagocytic process. In less than 2 h, foam cell morphology was acquired. The extent of loading obtained after 2 h was a linear function of the mass of cholesteryl oleate provided to the cells. The cholesteryl oleate incorporated was hydrolyzed in the cells at a linear rate over 24 h and the fractional hydrolysis was constant over a wide range of cellular esterified cholesterol contents. The rate of hydrolysis was influenced by the physical state of the cholesteryl ester; cholesteryl oleate in isotropic droplets was hydrolyzed 2-3-fold more rapidly than cholesteryl oleate in anisotropic droplets. The hydrolysis of both types of droplets was inhibited by lysosomotropic agents, indicating that hydrolysis occurred in the lysosomes. Only a small fraction (less than 10% after 24 h) of the free [3H]cholesterol generated in the lysosomes was esterified by ACAT resulting in a doubling of the cell free cholesterol content. Electron microscopy of cells treated with digitonin revealed the accumulation of free cholesterol in lipid-laden lysosomes. ACAT was active as endogenous free [14C]cholesterol was esterified in a linear manner over 24 h and was responsive to the presence of lysosomally-derived cholesterol, as the extent of esterification of the endogenous pool was directly proportional to the mass of [3H]cholesterol generated in the lysosomes.     

Protein kinase C potentiates isoproterenol-mediated cyclic AMP production without modifying the homologous desensitization process in J774 cells

The J774 murine macrophage cells possess a beta 2-adrenergic receptor coupled to adenylate cyclase, which can be regulated by homologous desensitization. Stimulation of protein kinase C by phorbol esters or oleoyl acetyl glycerol potentiates two-to-threefold the isoproterenol-induced cyclic AMP accumulation. These promoters act at a post-receptor level, since the number and affinity of the beta-adrenergic receptors, measured by use of the hydrophilic ligand [3H]CGP-12177, are not modified. In addition, the effect of cholera toxin is similarly increased and pretreatment of the cells with pertussis toxin prevents the action of phorbol esters. On the other hand, these promoters are ineffective on isoproterenol-induced desensitization and the rates of receptor segregation and recovery remain unchanged. Therefore, protein kinase C modulates the isoproterenol-stimulated adenylate cyclase, whereas it is inactive on the homologous desensitization process.     

Extracellular ATP4- promotes cation fluxes in the J774 mouse macrophage cell line

Extracellular ATP stimulates transmembrane ion fluxes in the mouse macrophage cell line J774. In the presence of Mg2+, nonhydrolyzable ATP analogs and other purine and pyrimidine nucleotides do not elicit this response, suggesting the presence of a specific receptor for ATP on the macrophage plasma membrane. One candidate for such a receptor is the ecto-ATPase expressed on these cells. We, therefore, investigated the role of this enzyme in ATP-induced 86Rb+ efflux in J774 cells. The ecto-ATPase had a broad nucleotide specificity and did not hydrolyze extracellular ATP in the absence of divalent cations. 86Rb+ efflux was not blocked by inhibition of the ecto-ATPase and did not require Ca2+ or Mg2+. In fact, ATP-stimulated 86Rb+ efflux was inhibited by Mg2+ and correlated with the availability of ATP4- in the medium. In the absence of divalent cations, the slowly hydrolyzable ATP analogs adenosine 5'-(beta, gamma-imido)triphosphate (AMP-PNP) and adenosine 5'-O-(3-thio)triphosphate (ATP-gamma-S) also stimulated 86Rb+ efflux, albeit at higher concentrations than that required for ATP4-. Exposure of J774 cells to 10 mM ATP for 45 min caused death of 95% of cells. By this means we selected variant J774 cells that did not exhibit 86Rb+ efflux in the presence of extracellular ATP but retained ecto-ATPase activity. These results show that the ecto-ATPase of J774 cells does not mediate the effects of ATP on these cells; that ATP4- and not MgATP2- promotes 86Rb+ efflux from these cells; and that hydrolysis of ATP is not required to effect this change in membrane permeability. These findings suggest that J774 cells possess a plasma membrane receptor which binds ATP4-, AMP-PNP, and ATP-gamma-S, and that the ecto-ATPase limits the effects of ATP on these cells by hydrolyzing Mg-ATP2-.     

Dibutyryl-cyclic AMP inhibits cholesterol esterification in J 774 monocyte-like cells

The effect of dibutyryl-cyclic AMP (dbcAMP) and theophylline was investigated on oleic acid incorporation into cholesteryl esters and triacylglycerols in the mouse monocyte-macrophage cell line J 774. 24h pretreatment of macrophages with dbcAMP decreased cholesteryl ester formation in a dose-dependent manner (about 4 fold reduction for dbcAMP 10(-4)M + theophylline 10(-3)M), while oleic acid incorporation into triacylglycerols was markedly (2 to 3 fold) enhanced. The catabolism of acetylated LDL was only slightly affected (about 15-20% reduction with dbcAMP 5 X 10(-4)M + theophylline 10(-3)M). Acyl Coenzyme A: cholesterol-O-acyl-transferase activity, measured in vitro on cell homogenates, was reduced in dbcAMP-treated cells, whereas diacylglycerol acyltransferase activity was increased. These results suggest that cyclic AMP can modulate cholesteryl ester and triacylglycerol formation in macrophages, and that these metabolisms are inversely regulated. Agents which increase cyclic AMP intracellular level could be of interest for reducing cholesteryl ester accumulation in macrophages.     

Control of the cell division cycle and sporulation in Saccharomyces cerevisiae by the cyclic AMP system

This paper reviews recent data on the adenylate cyclase system of the yeast Saccharomyces cerevisiae. Since the discovery of yeast adenylate cyclase mutants and the possibility of molecular biological analysis, adenylate cyclase and the subsequent steps in the cAMP cascade have become subject of intense investigation. CYR1, the structural gene for the adenylate cyclase catalytic subunit is necessary for cell division and in diploid cells is involved in the choice between sporulation and cell division. The cell division cycle in yeast is initiated by a step called START, which has been defined by mutations causing an arrest of the cells in an unbudded state. One class of mutation causes the cell to arrest at the same stage of the cell division cycle as the pheromone implicated in conjugation. A second class causes cells to cease growth in a different manner, but one which is similar to the arrest brought about by nutient deprivation. The adenylate cyclase gene belongs to the second class and has been identified as CDC35. Two genes of the first class have been cloned and sequenced. CDC28 codes for a kinase which has homology with the src proto-oncogene family. CDC36 is partly homologous with the oncogene ets. Two genes related to the ras oncogene family have also been implicated in the control of START. START can be dissociated in two subsequent phases, the first being controlled by the AMPc system and the second including proto-oncogenes. A model in which cAMP is a positive indicator of available nutrients such as nitrogen has been constructed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the acidification of endosomes and lysosomes by the antibiotic concanamycin B in macrophage J774.

The antibiotic concanamycin B was found to inhibit oxidized-low-density-lipoprotein(LDL)-induced accumulation of lipid droplets in the macrophage J774 at a concentration of 5-10 nM. Concanamycin B inhibited cholesteryl-ester synthesis from [14C]oleate by 50% at 14 nM without affecting the synthesis of triacylglycerol and polar lipids. Degradation of internalized oxidized 125I-LDL was inhibited by about 80% in cells treated with 25 nM concanamycin B, while cell-surface binding of oxidized 125I-LDL at 4 degrees C, uptake of surface-bound oxidized 125I-LDL and microsomal acyl-CoA:cholesterol acyltransferase activity were not significantly affected by the antibiotic at 25 nM. When J774 cells were treated with 25 nM concanamycin B at 37 degrees C for 60 min, there was a reduction of about 50% in the activity of cell-surface receptors. This reduction appeared to be due to partial trapping of the receptors within the cells. Concanamycin B significantly inhibited ATP-dependent acidification of endosomes and lysosomes of the J774 cells at a concentration of 4 nM. Since acidic condition of these organelles is required for receptor recycling and hydrolysis of lipoproteins, the results demonstrate that concanamycin-B inhibition of oxidized-LDL-induced accumulation of lipid droplets and cholesteryl esters in macrophages J774 is associated with reduced ATP-dependent acidification of these organelles.     

Control of growth and differentiation by cyclic AMP in fungi

InSaccharomyces cerivisiae intracellular cAMP mediates environmental signals that regulate cellular metabolism and growth. The studies on the cAMP-requiring mutants and their suppressors in the yeast revealed that cAMP-dependent protein phosphorylation is involved in the G1 phase of the cell cycle, stimulation of the phosphoinositide pathway and the post-meiotic stage of spourlation, and that inhibition of cAMP-dependent protein phosphorylation is required to go into the GO stage of and to induce meiotic division. Growth of some filamentous fungi was observed with significantly reduced levels of cAMP, suggesting that cAMP may not be essential for growth in some species of fungi. Germination of fungal spores, yeast-mycelium dimorphism and hyphal morphogenesis of several species of fungi were affected by cAMP. cAMP was involved in extension of hyphae, formation of hyphal aggregates and fruit body formation. Phosphorylation of cellular proteins is required in these processes, and the nature of these proteins phosphorylated by cAMP-dependent protein kinase is important to the understanding of the role of cAMP for growth and differentistion in fungal cells.     

Transient increase in cyclic AMP localized to macrophage phagosomes

Cyclic AMP (cAMP) regulates many biological processes and cellular functions. The importance of spatially localized intracellular gradients of cAMP is increasingly appreciated. Previous work in macrophages has shown that cAMP is produced during phagocytosis and that elevated cAMP levels suppress host defense functions, including generation of proinflammatory mediators, phagocytosis and killing. However, the spatial and kinetic characteristics of cAMP generation in phagocytosing macrophages have yet to be examined. Using a Förster resonance energy transfer (FRET)-based cAMP biosensor, we measured the generation of cAMP in live macrophages. We detected no difference in bulk intracellular cAMP levels between resting cells and cells actively phagocytosing IgG-opsonized particles. However, analysis with the biosensor revealed a rapid decrease in FRET signal corresponding to a transient burst of cAMP production localized to the forming phagosome. cAMP levels returned to baseline after the particle was internalized. These studies indicate that localized increases in cAMP accompany phagosome formation and provide a framework for a more complete understanding of how cAMP regulates macrophage host defense functions.     

Regulation of microtubule assembly and disassembly by nucleotides

The role of nucleotides in microtubular assembly and disassembly has been reviewed. Two possible functions of GTP hydrolysis during assembly are discussed: (1) hydrolysis renders sensitivity to factor(s) regulating microtubule depolymerization; (2) the energy of GTP hydrolysis is utilized for the subunit flow from one end of the microtubule to the other. In the second part of the review, experiments are considered showing that microtubular disassembly takes place in the cells only in the presence of ATP, and, therefore, this process is regulated via some ATP-dependent mechanism (most probably, phosphorylation of microtubule-associated proteins).     

Dose-dependent dual effects of cholesterol and desmosterol on J774 macrophage proliferation

We addressed the ability of native, oxidized and acetylated low-density lipoproteins (nLDL, oxLDL and acLDL, respectively) and desmosterol to act as sources of sterol for the proliferation of J774A.1 macrophages. Treatment with 0.5 μM lovastatin and lipoprotein-deficient serum suppressed cell proliferation. This inhibition was effectively prevented by nLDL, but only to a lesser extent by oxLDL. AcLDL, despite its ability to deliver a higher amount of cholesterol to J774 macrophages than the other LDLs, was dependent on mevalonate supply to sustain cell proliferation. Similarly, exogenous desmosterol, which is not converted into cholesterol in J774 cells, required the simultaneous addition of mevalonate to support optimal cell growth. Expression of hydroxymethyl glutaryl coenzyme A reductase mRNA was potently down-regulated by acLDL and exogenous desmosterol, but the effect was weaker with other sterol sources. We conclude that nLDL is more efficient than modified LDL in sustaining macrophage proliferation. Despite the requirement of cholesterol or desmosterol for J774 cell proliferation, excessive provision of either sterol limits mevalonate availability, thus suppressing cell proliferation.     

Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly

Cross talk between the actin cytoskeleton and the microtubule (MT) network plays a critical role in regulation of endothelial permeability. We have previously demonstrated that MT disruption by nocodazole results in increases in MLC phosphorylation, actomyosin contraction, cell retraction, and paracellular gap formation, cardinal features of endothelial barrier dysfunction (Verin AD, Birukova A, Wang P, Liu F, Becker P, Birukov K, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 281: L565-L574, 2001; Birukova AA, Smurova K, Birukov KG, Usatyuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva A, Garcia JG, and Verin AD. J Cell Physiol. In press.). Although activation of PKA opposes barrier-disrupting effects of edemagenic agents on confluent EC monolayers, information about the molecular mechanisms of PKA-mediated EC barrier protection is limited. Our results suggest that MT disassembly alters neither intracellular cAMP levels nor PKA enzymatic activity; however, elevation of cAMP levels and PKA activation by either cholera toxin or forskolin dramatically attenuates the decline in transendothelial electrical resistance induced by nocodazole in human pulmonary EC. Barrier-protective effects of PKA on EC were associated with PKA-mediated inhibition of nocodazole-induced stress fiber formation, Rho activation, phosphorylation of myosin phosphatase regulatory subunit at Thr696, and decreased MLC phosphorylation. In addition, forskolin pretreatment attenuated MT disassembly induced by nocodazole. These results suggest a critical role for PKA activity in stabilization of MT cytoskeleton and provide a novel mechanism for cAMP-mediated regulation of Rho-induced actin cytoskeletal remodeling, actomyosin contraction, and EC barrier dysfunction induced by MT disassembly.     

The modulation of transcobalamin II (TC-II) production by cyclic adenosine 3',5'-monophosphate in the murine macrophage cell line J774: relationship to growth behavior

We undertook a study to define the role of cyclic AMP [cAMP] in modulating the secretion of transcobalamin II (TC-II) in the mouse macrophage like cell line J774. J774 was observed to secrete large amounts of TC-II, particularly in the presence of 8-bromo cAMP or cholera toxin or when grown in medium supplemented with low concentrations of horse serum (1% or 5%) or in serum-free medium. Variant cell lines derived from J774 and deficient either in adenylate cyclase (ac -) or cAMP-dependent protein kinase (pk -) activity showed very low and intermediate levels of basal secretory activity of TC-II, respectively, compared to J774. Maximum secretory activity of TC-II was observed in J774 under conditions in which growth was poorest (in the presence of 8-bromo-cAMP or 1% or 5% horse serum-supplemented medium or in serum-free medium). Cells grown in serum-free medium were found to have elevated basal adenylate cyclase activity and cAMP levels compared to those grown in medium supplemented with 20% horse serum. The data from this study demonstrate a negative correlation between growth activity and TC-II secretion in the J774 cell line. The stimulatory effect of exogenous cAMP on TC-II secretion by J774, the reduced secretory activity of the variant lines ac- and pk- and the observed increase in cell cAMP levels under conditions of serum starvation in which TC-II secretion is considerably enhanced, suggest that cell cAMP is an important modulator of TC-II secretion and growth behavior in the J774 cell line.     

Ionophore-induced disassembly of blood platelet microtubules: effect of cyclic AMP and indomethacin

Cytoplasmic calcium levels are believed to be important in blood platelet activation. Upon activation, the discrete marginal microtubule band, which maintains the discoid shape of non-activated platelets, becomes disrupted. Present studies demonstrate that the extent of assembly of the marginal microtubule band is related to cytoplasmic calcium levels. The divalent cationophore, A23187, causes platelet aggregation, secretion, and contraction by promoting calcium transport from intraplatelet storage sites into the cytoplasm. A23187 caused disassembly of platelet microtubules. Quantitation of electron micrographs revealed that numbers of microtubules were reduced by approximately 80% after A23187 treatment. Secondly, assembled microtubules in homogenates of platelets, in which microtubules were stabilized prior to homogenization, were decreased in favor of free tubulin in A23187-treated platelets. Thirdly, A23187 increased 14C-colchicine binding by intact platelets; this also indicated a shift in the microtubule subunit equilibrium to favor free, colchicine-binding tubulin subunits. In control experiments, A23187 did not affect the stability of platelet tubulin, the colchicine binding reaction, or the total tubulin content of platelets. Stimulation of colchicine binding depended on A23187 concentration (0.05-0.5 microM) and did not require extracellular calcium. A23187-stimulation of colchicine binding was blocked by dibutyryl cyclic AMP (0.80 mM) and/or 3-isobutyl-1-methylxanthine (50 microM) and by indomethacin (10 microM). Cyclic AMP or indomethacin also interferes with A23187-induced platelet activation, but indomethacin is not likely to completely inhibit the perturbation of intraplatelet calcium gradients by A23187. It is suggested that A23187-induced microtubule disassembly may be an indirect effect of calcium on microtubules.     

Inhibition of the accumulation of lipid droplets in macrophage J774 by bafilomycin B1 and destruxin E.

Two microbial metabolites, bafilomycin B1 and destruxin E, have been found to inhibit significantly the oxidized low density lipoprotein (LDL)-induced accumulation of lipid droplets at 3 nM and 0.5 microM, respectively, in macrophage J774. The incorporation of [14C]oleate into cholesteryl esters in the cells incubated with oxidized LDL was inhibited to the same extent by the two compounds. Both compounds had no effect on the cell surface binding at 4 degrees C and the internalization of oxidized 125I-LDL as well as on the activity of acyl-CoA:cholesterol acyltransferase. However, when incubated with these compounds at 37 degrees C, receptors for oxidized LDL were partially trapped within the cell. In accordance with receptor accumulation, ATP-dependent acidification of endosomes and lysosomes was significantly inhibited by 50 nM bafilomycin B1 and 1 microM destruxin E, respectively. From these results it was concluded that the inhibition of ATP-dependent acidification of endosomes and lysosomes by bafilomycin B1 and destruxin E resulted in the reduction of oxidized LDL-induced synthesis of cholesteryl ester and thereby caused a reduced accumulation of lipid droplets in macrophage J774.     

Enumeration of phagocytosed Staphylococcus aureus inside cells of the mouse macrophage cell line J774 by bioluminescence,fluorescence and viable counting techniques

In order to quantify intracellular Staphylococcus aureus within a macrophage-like cell line by a bioluminescence technique, the mouse cell line J774 and opsonized Staphylococcus aureus were incubated together to allow phagocytosis to occur. Experiments using UV microscopy and fluorescent stained S. aureus were performed to determine an estimate of the mean intracellular bacterial numbers. For enumeration of intracellular bacteria by a bioluminescence technique, extracellular bacteria were removed by washing, the macrophages lysed mechanically and osmotically and treated with apyrase to remove somatic ATP. Bacterial cells were washed and the intracellular ATP measured by firefly luciferase bioluminescence in a luminometer. This new method of enumerating intracellular bacteria was compared to the conventional method of viable counts and found to correlate (r = 0.78). The bioluminescence assay developed was found to be a relatively rapid alternative method to the techniques currently used to enumerate intracellular bacteria and could prove advantageous in studies of intracellular killing and effects of antimicrobial agents on intracellular pathogens.     

Cholesterol esterification as a limiting factor in accumulation of cell cholesterol: a comparison of two J774 macrophage cell lines

It was previously reported by Tabas et al. that J774 macrophages, unlike mouse peritoneal macrophages, accumulate large amounts of cholesteryl esters when incubated with native low-density lipoprotein (LDL). Comparison of the cell line (designated J774A.2) used in those experiments with its parent line (J774A.1) indicates that it is a variant with a greater rate of cholesterol esterification. This large difference in cholesterol esterification was accompanied by only a small difference in rates of LDL uptake and degradation by the J774A.2 line. The J774A.2 cells have become a variant line through either mutation or selection which has enhanced its susceptibility to foam cell formation by its markedly increased ability to esterify cholesterol.     

Antioxidant status in J774A.1 macrophage cell line during chronic exposure to glycated serum.

Advanced glycation end-products (AGEs) are linked to aging and correlated diseases. The aim of present study was to evaluate oxidative stress related parameters in J774A.1 murine macrophage cells during chronic exposure to a subtoxic concentration of AGE (5% ribose-glycated serum (GS)) and subsequently for 48 h to a higher dose (10% GS). No effects on cell viability were evident in either experimental condition. During chronic treatment, glycative markers (free and bound pentosidine) increased significantly in intra- and extracellular environments, but the production and release of thiobarbituric acid reactive substances (TBARs), as an index of lipid peroxidation, underwent a time-dependent decrease. Exposure to 10% GS evidenced that glycative markers rose further, while TBARs elicited a cellular defence against oxidative stress. Nonadapted cultures showed an accumulation of AGEs, a marked oxidative stress, and a loss of viability. During 10% GS exposure, reduced glutathione levels in adapted cultures remained constant, as did the oxidized glutathione to reduced glutathione ratio, while nonadapted cells showed a markedly increased redox ratio. A constant increase of heat shock protein 70 (HSP70) mRNA was observed in all experimental conditions. On the contrary, HSP70 expression became undetectable for a longer exposure time; this could be due to the direct involvement of HSP70 in the refolding of damaged proteins. Our findings suggest an adaptive response of macrophages to subtoxic doses of AGE, which could constitute an important factor in the spread of damage to other cellular types during aging.