Taurine chloramine and taurine bromamine induce heme oxygenase-1 in resting and LPS-stimulated J774.2 macrophages

Summary. Taurine chloramine (TauCl) and taurine bromamine (TauBr) are products of activated neutrophils and eosinophils, respectively. It has been reported that TauCl, has strong anti-inflammatory properties. In a number of separate studies it has been shown that heme oxygenase-1 (HO-1), a stress inducible protein, exerts similar anti-inflammatory effects. In this study we investigated the influence of HO-1 on TauCl/TauBr mediated suppression of NO generation in J774.2 macrophages. Expression of HO-1 and inducible nitric oxide synthase (NOS-2) in LPS stimulated J774.2 cells provides an opportunity for determining these interactions. TauCl and TauBr, at non-cytotoxic concentrations, in a similar, dose-dependent manner, inhibited the expression of NOS-2, as evidenced by western blotting technique. Surprisingly, TauCl and TauBr induced expression of HO-1 in both non-activated and LPS-activated macrophages. Importantly, the fall in NOS-2 protein level was associated with a concomitant, dose-dependent induction of HO-1. In addition, an inhibitor of HO-1 activity, chromium III mesoporhyrin (CrMP), attenuated the inhibitory activity of TauBr but not that of TauCl, as measured by nitrite accumulation. These results suggest that at a site of inflammation, TauCl and TauBr may provide a link between taurine-dependent and HO-1-dependent cytoprotective mechanisms.     

Tubular lysosomes accompany stimulated pinocytosis in macrophages

A network of tubular lysosomes extends through the cytoplasm of J774.2 macrophages and phorbol ester-treated mouse peritoneal macrophages. The presence of this network is dependent upon the integrity of cytoplasmic microtubules and correlates with high cellular rates of accumulation of Lucifer Yellow (LY), a marker of fluid phase pinocytosis. We tested the hypothesis that the efficiency of LY transfer between the pinosomal and lysosomal compartments is increased in the presence of tubular lysosomes by asking how conditions that deplete the tubular lysosome network affect pinocytic accumulation of LY. Tubular lysosomes were disassembled in cells treated with microtubule-depolymerizing drugs or in cells that had phagocytosed latex beads. In unstimulated peritoneal macrophages, which normally contain few tubular lysosomes and which exhibit relatively inefficient transfer of pinocytosed LY to lysosomes, such treatments had little effect on pinocytosis. However, in J774 macrophages and phorbol ester-stimulated peritoneal macrophages, these treatments markedly reduced the efficiency of pinocytic accumulation of LY. We conclude that a basal level of solute accumulation via pinocytosis proceeds independently of the tubular lysosomes, and that an extended tubular lysosomal network contributes to the elevated rates of solute accumulation that accompany macrophage stimulation. Moreover, we suggest that the transformed mouse macrophage cell line J774 exhibits this stimulated pinocytosis constitutively.     

Phagocytosis of antibody‐opsonized tumor cells leads to the formation of a discrete vacuolar compartment in macrophages

Despite the rapidly expanding use of antibody‐based therapeutics to treat cancer, knowledge of the cellular processes following phagocytosis of antibody‐opsonized tumor cells is limited. Here we report the formation of a phagosome‐associated vacuole that is observed in macrophages as these degradative compartments mature following phagocytosis of HER2‐positive cancer cells in the presence of the HER2‐specific antibody, trastuzumab. We demonstrate that this vacuole is a distinct organelle that is closely apposed to the phagosome. Furthermore, the size of the phagosome‐associated vacuole is increased by inhibition of the mTOR pathway. Collectively, the identification of this vacuolar compartment has implications for understanding the subcellular trafficking processes leading to the destruction of phagocytosed, antibody‐opsonized cancer cells by macrophages.      

Effect of molecular size of 125I-labelled poly(vinylpyrrolidone) on its pinocytosis by rat visceral yolk sacs and rat peritoneal macrophages.

Rates of pinocytosis of different molecular-weight distributions of 125I-labelled poly(vinylpyrrolidone) by rat visceral yolk sacs and rat peritoneal macrophages were measured in vitro. Four preparations of mean molecular weights 50 000, 84 000, 700 000 and 7 000 000, were used. Macrophages captured the highest-molecular-weight preparation more rapidly than the other preparations. In contrast, rate of capture by the yolk sac decreased with increasing molecular weight. Incubations with a very-high-molecular-weight fraction derived from the 7 000 000-average-mol. wt. preparation clearly demonstrated that very large polymer molecules are not accumulated by the yolk sac, but are preferentially captured by macrophages. Analysis of the 125I-labelled poly(vinylpyrrolidone) internalized by the two cell types confirmed that low-molecular-weight material is preferred by the yolk sac, whereas the macrophage is less discriminating.     

The effects of acetylated low-density lipoproteins on fluid-phase pinocytosis by macrophages

A simple method has been set up to measure the rate of fluid-phase pinocytosis in resident mouse peritoneal macrophages in culture. The method uses 125I-labelled polyvinylpyrrolidone as a nondegradable marker of fluid-phase pinocytosis. The accumulation of 125I-labelled polyvinylpyrrolidone by the cells was directly proportional to its concentration in the culture medium up to at least 200 micrograms/ml. The estimates of the rate of fluid-phase pinocytosis were reproducible within each experiment (coefficient of variation 8.5%) but varied between individual experiments. Fluid-phase pinocytosis was undetectable at 4 degrees C and reduced greatly at 37 degrees C by metabolic inhibitors and 1 mM ZnSO4. High concentrations of human acetylated low-density lipoproteins, which are taken up rapidly by macrophages, decreased the rate of fluid-phase pinocytosis by up to about 70%. The inhibition was seen after only 2 h of incubation. Unmodified low-density lipoproteins, which are taken up only slowly by macrophages, did not usually inhibit fluid-phase pinocytosis (in fact, they sometimes increased it). Modified low-density lipoprotein uptake, leading to massive lipid accumulation in macrophages in the arterial wall, has been postulated to be involved in the pathogenesis of atherosclerosis. This study raises the possibility that the rate of fluid-phase pinocytosis in these lipid-laden arterial macrophages may be reduced.     

A prelysosomal compartment sequesters membrane-impermeant fluorescent dyes from the cytoplasmic matrix of J774 macrophages

After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm.     

Fluorescent reporter proteins for the tonoplast and the vacuolar lumen identify a single vacuolar compartment in Arabidopsis cells

We generated fusions between three Arabidopsis (Arabidopsis thaliana) tonoplast intrinsic proteins (TIPs; alpha-, gamma-, and delta-TIP) and yellow fluorescent protein (YFP). We also produced soluble reporters consisting of the monomeric red fluorescent protein (RFP) and either the C-terminal vacuolar sorting signal of phaseolin or the sequence-specific sorting signal of proricin. In transgenic Arabidopsis leaves, mature roots, and root tips, all TIP fusions localized to the tonoplast of the central vacuole and both of the lumenal RFP reporters were found within TIP-delimited vacuoles. In embryos from developing, mature, and germinating seeds, all three TIPs localized to the tonoplast of protein storage vacuoles. To determine the temporal TIP expression patterns and to rule out mistargeting due to overexpression, we generated plants expressing YFP fused to the complete genomic sequences of the three TIP isoforms. In transgenic Arabidopsis, gamma-TIP expression was limited to vegetative tissues, but specifically excluded from root tips, whereas alpha-TIP was exclusively expressed during seed maturation. delta-TIP was expressed in vegetative tissues, but not root tips, at a later stage than gamma-TIP. Our findings indicate that, in the Arabidopsis tissues analyzed, two different vacuolar sorting signals target soluble proteins to a single vacuolar location. Moreover, TIP isoform distribution is tissue and development specific, rather than organelle specific.     

Taxol induces the formation of unusual arrays of cellular microtubules in colchicine-pretreated J774.2 cells

Taxol is an antimitotic agent with the unique ability to induce the formation of parallel arrays of microtubules in cells. We have studied the effects of taxol on microtubule organization in the cultured macrophage-like cell line, J774.2, and shown that this novel reorganization of cellular microtubules is both a concentration-dependent and time-dependent phenomenon. In this paper, we have examined in detail the unusual microtubule arrays induced by taxol in colchicine-pretreated cells. Interphase cells which are pretreated with the irreversible inhibitor, colchicine, and then treated with taxol form a single microtubule aster associated with the nucleus and numerous discrete sites of apparent microtubule nucleation scattered throughout the cytoplasm. One interesting possibility is that these structures represent nucleation sites for taxol-induced bundles, a result supporting the notion that taxol-induced microtubule arrays are organized assemblies at what are perhaps secondary organizing sites.     

Effect of suramin on pinocytosis by resident rat peritoneal macrophages: an analysis using four different substrates

The effect of suramin on pinocytosis and intralysosomal proteolysis by resident rat peritoneal macrophages cultured in vitro has been studied. Suramin had little effect on the rate of pinocytic uptake of two non-adsorptive substrates [14C]sucrose and [3H]dextran, but unexpectedly enhanced uptake of a third, 125I-labelled polyvinylpyrrolidone (PVP). Since this enhanced uptake was completely abolished by NaF at a concentration known to inhibit pinocytosis, it clearly represented an increased internalization of substrate and not merely a superficial binding to the cell surface. It was concluded that suramin (i) does not affect the rate of formation of pinocytic vesicles but (ii) acts as a bivalent ligand, binding to both the macrophage surface and the 125I-labelled polyvinylpyrrolidone, thus converting a non-adsorptive into an adsorptive substrate. Suramin (500 micrograms/ml) decreased both the rate of uptake of formaldehyde-denatured 125I-labelled bovine serum albumin (BSA) (an adsorptive substrate) and the rate of its subsequent intracellular degradation. Thus, depending on the substrate chosen to measure pinocytosis, the same modifier may stimulate or inhibit uptake or be without effect.     

Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae

Intact vacuoles are released from spheroplasts of Saccharomyces cerevisiae by means of a gentle mechanical disintegration method. They are purified by centrifugation in isotonic density gradients (flotation and subsequent sedimentation), and analyzed for their soluble amino acid content. The results indicate that about 60% of the total amino acid pool of spheroplasts is contained in the vacuoles. This may be an underestimate, as it presupposes no loss of amino acids from the vacuoles during the purification procedure. The amino acid concentration in the vecuoles is calculated to be approximately 5 times that in the cytoplasm if the total volumes of the two compartments are used for the calculation. The vacuolar amino acid pool is rich in basic amino acids, and in citrulline and glutamine, but contains a remarkably small amount of glutamate. Radioactive labeling experiments with spheroplasts indicate that the vacuolar amino acids are separated from the metabolically active pools located in the cytoplasm. This is particularly evident for the basic amino acids and glutamine; in contrast, the neutral amino acids and glutamate appear to exchange more rapidly between the cytoplasmic and the vacuolar compartments of the cells.     

Analysis of the effect of amines on inhibition of receptor-mediated and fluid-phase pinocytosis in rabbit alveolar macrophages

Incubation of rabbit alveolar macrophages in vitro with methyl amine led to a time- and concentration-dependent inhibition of uptake of alpha macroglobulin-125I-trypsin complexes (alpha M-125I-T). Upon addition of methyl amine (50 mM) to cells prelabeled with alpha M-125I-T there was a rapid inhibition of lysosomal catabolism of internalized ligand. In the absence of ligand, incubation of cells with 50 mM methyl amine led to a 40-70% decrease in surface-receptor number. The methyl amine-induced decrease in surface-receptor number only occurred in metabolically active cells since cells incubated at 0 degrees C, or treated with N-ethyl maleimide and incubated at 37 degrees C, did not show the effect. Incubation of cells at 37 degrees C with methyl amine also effected a 40-70% decrease in fluid-phase pinocytosis. Although there was a decline in surface-receptor number, the remaining population of receptors were capable of mediating (at least) one round of ligand internalization. However, further ligand uptake was prevented. Data demonstrate that although receptors were present on cell surfaces, they were incapable of mediating ligand internalization. Incubation of macrophages with chloroquine at 37 degrees C for 60 min also led to a disappearance of receptors, and a concomitant reduction in fluid-phase pinocytosis.     

Expression of phenotypic traits following modulation of colchicine resistance in J774.2 cells

Development of resistance to colchicine in the mouse macrophage-like cell line J774.2 coincides with the expression of a variety of phenotypic traits. A cloned subline (J7/CLC-20), maintained in 20 microM colchicine, exhibits reduced steady-state association with drug, increased presence of a 140,000-145,000 dalton (140-145 kD) phosphoglycoprotein associated with the plasma membrane, double minute chromosomes and cross-resistance to other drugs. While similar phenotypic traits are observed in J774.2 cells resistant to taxol and vinblastine, differences in the electrophoretic mobilities of the resistance-specific glycoproteins in each of the three sublines suggest that multi-drug resistant sublines exhibit specificity for individual drugs. In an attempt to elucidate the relationships between the phenotypic traits associated with colchicine resistance, the degree of colchicine resistance in J7/CLC-20 cells was modulated and the levels of expression of the phenotypic traits were quantitated. In the absence of colchicine in the growth medium, J7/CLC-20 cells reverted to drug sensitivity within 35 days. A decrease in the level of resistance coincided with coordinate changes in both the quantity of the resistance-specific glycoprotein and the average number of double minute chromosomes. We propose that the emergence and disappearance of the resistance-specific glycoprotein and double minute chromosomes may be closely linked. However, J7/CLC-20 cells which had regained their drug sensitivity after growth in drug-free medium maintained a reduced level of steady-state drug association. The persistence of reduced drug association in cells that have reverted to a drug-sensitive state suggests that this phenomenon, although related to colchicine resistance, need not be the primary or only mechanism of drug resistance.     

Use of antibodies to probe membrane glycoproteins associated with drug-resistant J774.2 cells

Three drug-resistant sublines of the murine macrophage-like cell line J774.2 were selected in vitro for their ability to grow in high concentrations of either taxol, vinblastine, or colchicine. Each contains a major plasma membrane glycoprotein (130-150 kDa), which is barely seen in the drug-sensitive parental cell line. Polyclonal antibodies, raised against the glycoproteins present in the colchicine- and vinblastine-resistant cells, were used to probe for relationships among the three glycoproteins. Our observations suggest that the glycoproteins from the different drug-resistant cell lines share many common domains but are not identical.     

Mechanism underlying oxidative stress-mediated lipotoxicity: exposure of J774.2 macrophages to triacylglycerols facilitates mitochondrial reactive oxygen species production and cellular necrosis

The aim of this study was to elucidate death pathways in macrophages resulting from exposure to triacylglycerols (TG), mechanisms which may be relevant to the development of atherosclerosis. A commercial TG emulsion (lipid emulsion, LE; 0.1-1.5 mg lipids/ml) was added to J774.2 cells in culture. Within the first 24 h after TG treatment, cellular reactive oxygen species (ROS) levels were strongly elevated and basal caspase-3 activity was attenuated. In contrast, after 48 h, ROS production was arrested. TG-mediated ROS production was demonstrated to be via mitochondrial complex 1 of the electron-transfer chain since the inhibitor of complex 1 rotenone significantly attenuated the cellular ROS levels in TG-treated cells. The TG effect culminated in cell death, with no caspase-3 activation. We therefore evaluated the effect of TG on apoptotic cells showing high caspase activity. TG induced elevated ROS levels and suppressed caspase-3 in apoptotic cells pretreated for 24 h with cycloheximide. Dual staining with propidium iodide and Annexin V followed by flow cytometric analysis showed that TG facilitated cell death with clear necrotic characteristics. To elucidate whether the necrotic cell death process is indeed oxidant dependent, antioxidant protection was studied. Treatment with N-acetylcysteine (NAC) (0.5 mM), ascorbic acid (0.5 mM), and resveratrol (0.2 mM) protected against the TG lipotoxic effect, while, surprisingly, lipophilic antioxidants did not. The combination of NAC, ascorbic acid, and resveratrol, each at much lower concentrations, had a synergistic protective effect. In conclusion, we show here for the first time that exposure to TG can directly regulate lipotoxicity in macrophages by inducing mitochondria-mediated prolonged oxidative stress; this, in turn, can inactivate the apoptotic caspase system, resulting in necrotic cell death which can be prevented by specific antioxidants.     

Phorbol myristate acetate stimulates pinocytosis and membrane spreading in mouse peritoneal macrophages

Phorbol myristate acetate (PMA) at a concentration of 0.01 microgram/ml causes an approximately threefold increase in surface area of resident, proteose-peptone-elicited, and thioglycolate-broth-elicited mouse peritoneal macrophages. Resident and proteose-peptone-elicited macrophages, cultured for 24 h in the presence of PMA, increase their pinocytic rate twofold in response to addition of PMA (0.01 microgram/ml) to the medium. Thioglycolate-broth-elicited macrophages, cultured for 24 h in the absence of PMA, immediately increase their pinocytic rate 2- to 3.5-fold in response to a single challenge with PMA (0.01 microgram/ml). Cytochalasin B, colchicine, and podophyllotoxin have only modest inhibitory effects on the basal rate of pinocytosis and on PMA-induced cellular spreading, but completely block the stimulatory effects of PMA on pinocytosis in thioglycolate- broth-elicited macrophages. Cytochalasin D markedly inhibits both basal and PMA-stimulated pinocytosis in these cells. Thus, PMA is a useful tool for studying mechanisms of macrophage spreading and for enhancing the overall rate of pinosome formation.     

A quantitative study of pinocytosis and intracellular proteolysis in rat peritoneal macrophages.

A method for the culture of rat peritoneal macrophages in vitro is described, in which pinocytic uptake of colloidal [198 Au]gold, 125I--labelled poly(vinylpyrrolidone) and [14C]sucrose proceeds at contant and fairly reproducible rates for several hours. The rat of uptake of colloidal [198 Au]gold, which wxhibited some inter-batch variation, was approx. 100 times that of the other two substrates. Colloidal gold did not affect the rate of uptake of 125I-labelled poly(vinylpyrrolidone) and therefore its own high rate of uptake could not be attributed to a stimulation of the formation of pinocytic vesicles. It conclude that uptake of collodial gold is highly dependent on adsorption on binding sites on the plasma membrane. Uptake of formaldehyde-treated 125I-labelled bovine serum albumin was followed by the release of [125I]iodo-L-tyrosine into the culture medium and took place at a rate intermediate between those of collodial [198Au]gold and the other two non-digestible substrates, 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose.