Electronegative LDL induction of apoptosis in macrophages: Involvement of Nrf2

The aim of this study was to determine the apoptotic pathways and mechanisms involved in electronegative LDL [LDL(−)]-induced apoptosis in RAW 264.7 macrophages and the role of Nrf2 in this process. Incubation of RAW 264.7 macrophages with LDL(−) for 24 h resulted in dose-dependent cell death. Activated caspases were shown to be involved in the apoptosis induced by LDL(−); incubation with the broad caspase inhibitor z-VAD prevented apoptosis in LDL(−)-treated cells. CD95 (Fas), CD95 ligand (FasL), CD36 and the tumor necrosis factor (TNF) ligand Tnfsf10 were overexpressed in LDL(−)-treated cells. However, Bax, Bcl-2 and Mcl-1 protein levels remained unchanged after LDL(−) treatment. LDL(−) promoted hyperpolarization of the mitochondrial membrane, elevated reactive oxygen species (ROS) production and translocation of Nrf2 to the nucleus, a process absent in cells treated with native LDL. Elicited peritoneal macrophages from Nrf2-deficient mice exhibited an elevated apoptotic response after challenge with LDL(−), together with an increase in the production of ROS in the absence of alterations in CD36 expression. These results provide evidence that CD36 expression induced by LDL(−) is Nrf2-dependent. Also, it was demonstrated that Nrf2 acts as a compensatory mechanism of LDL(−)-induced apoptosis in macrophages.     

Metabolism of oxidized LDL by macrophages

Oxidation products of lipids and proteins are found in atherosclerotic plaque and in macrophage foam cells. Macrophages avidly endocytose in-vitro oxidized LDL and accumulate sterols. What is the evidence that such a process is involved in in-vivo foam cell formation? The present review surveys current knowledge on the metabolism of oxidized LDL by macrophages, and the types, amounts and location of oxidation products that accumulate in these cells. Comparable studies of lesion lipoproteins and foam cells indicate that limited extracellular lipoprotein oxidation, perhaps followed by more extensive intracellular oxidation subsequent to uptake by macrophages, is a more likely scenario in vivo.     

Integrating macrophages into organotypic co-cultures: a 3D in vitro model to study tumor-associated macrophages

Tumor progression is controlled by signals from cellular and extra-cellular microenvironment including stromal cells and the extracellular matrix. Consequently, three-dimensional in vitro tumor models are essential to study the interaction of tumor cells with their microenvironment appropriately in a biologically relevant manner. We have previously used organotypic co-cultures to analyze the malignant growth of human squamous cell carcinoma (SCC) cell lines on a stromal equivalent in vitro. In this model, SCC cell lines are grown on a collagen-I gel containing fibroblasts. Since macrophages play a critical role in the progression of many tumor types, we now have expanded this model by integrating macrophages into the collagen gel of these organotypic tumor co-cultures. This model was established as a murine and a human system of skin SCCs. The effect of macrophages on tumor progression depends on their polarization. We demonstrate that macrophage polarization in organotypic co-cultures can be modulated towards and M1 or an M2 phenotype by adding recombinant IFN-γ and LPS or IL-4 respectively to the growth medium. IL-4 stimulation of macrophage-containing cultures resulted in enhanced tumor cell invasion evidenced by degradation of the basement membrane, enhanced collagenolytic activity and increased MMP-2 and MMP-9. Interestingly, extended co-culture with tumor cells for three weeks resulted in spontaneous M2 polarization of macrophages without IL-4 treatment. Thus, we demonstrate that macrophages can be successfully integrated into organotypic co-cultures of murine or human skin SCCs and that this model can be exploited to analyze macrophage activation towards a tumor supporting phenotype.     

Zinc deficiency does not enhance LDL uptake by P 388 D1 macrophages in vitro

The aim of the study was to investigate the effect of zinc depletion on the susceptibility of Wistar rat low-density lipoproteins (LDL) to peroxidation and their uptake by macrophages, before and after in vitro oxidation. The rats were fed for 7 wk a Zn-adequate diet (100 ppm) ad libitum (AL), a Zn-deficient diet (0.2 ppm) ad libitum (ZD), or a Zn-adequate diet according to the pair-feeding method (PF). Zinc status was determined and, for each group, blood was pooled, and LDL were isolated and labeled with¹²⁵Iodine. An aliquot of each LDL sample was oxidized using Fe^II 10 μM/ascorbate 250 μM. Oxidized and nonoxidized (native) LDL were incubated with P 388 D1 macrophages, and their rates of uptake and degradation by macrophages were measured. Before oxidation, LDL uptake and degradation were not modified by the diet, suggesting that Zn deficiency did not modify rat LDL in vivo. After oxidation, both LDL uptake and degradation were significantly enhanced in the three groups. Nevertheless, we did not observe a significant effect of Zn deficiency. This observation suggests that, in our experimental conditions, Zn deficiency did not modify LDL catabolism.     

Silica toxicity for macrophages in vitro: a new cytotoxicity test.

Soluble factors released from silica-damaged macrophages inhibit proliferation of various haematopoietic cells in long term cultures. The same holds true for damage induced by heat, non-physiological pH, freezing and thawing. This phenomenon is dose-dependent and correlates with the degree of macrophage viability. Thus, a base for measuring the amount of damage to the macrophage is established.     

Decreased phagocytosis by peritoneal macrophages from BCG-treated mice: induction of the phagocytic defect in normal macrophages with BCG in vitro.

Mice treated up to 31 weeks previously with intraperitoneal BCG yielded peritoneal macrophages with decreased phagocytosis of starch granules, latex beads, graphite dust and formalinized Listeria monocytogenes, with or without opsonin, compared to macrophages from untreated mice. These assays were selected to allow quantitative determinations of the rate or extent of particle uptake under nonrate-limiting conditions. Phagocytosis could be depressed to a similar degree by the prior addition of either starch granules or BCG to normal adherent peritoneal cell cultures in vitro. However, with these two particles, two different mechanisms of inhibition of subsequent phagocytosis appeared to be at work. Inhibition of phagocytosis by prior exposure to large amounts of starch granules appeared to consist largely of mechanical interference, as if through the preemption of intracellular space. In contrast, inhibition by prior uptake of BCG occurred with very small amounts of BCG and appeared gradually with time after uptake of BCG. The ability of a “macrophage activating agent” to inhibit selected functions of parasitized cells may help to explain some of the discordant results obtained by others studying phagocytosis by “activated” macrophages. Such agents may simultaneously enhance some macrophage functions and depress others.     

In vitro and in vivo induction of nitric oxide by murine macrophages stimulated with Bordetella pertussis

Abstract Nitric oxide (NO) exhibits potent antimicrobial activity in vitro. The function of NO in host defenses in vivo, however, is presently unclear. Experiments were undertaken to determine the production of NO in vitro from murine peritoneal and alveolar macrophages, and murine macrophage cell line (J774A.1) stimulated with Bordetella pertussis or pertussis toxin (PT). In addition, we determined circulating levels of NO in the sera and bronchoalveolar lavage (BAL) fluids of mice infected intranasally with B. pertussis . The results of this study showed that in vitro murine peritoneal macrophages induce production of NO in response to B. pertussis and PT. In addition, murine macrophage cell line, J774A.1 also induces NO production after stimulation with B. pertussis . NO production was also detected in alveolar macrophages from mice infected intranasally with B. pertussis . Finally, a significant increment of circulating levels of NO was noted, in the sera but not in the BAL fluids, of mice infected intranasally with B. pertussis .     

Sequestration of aggregated LDL by macrophages studied with freeze-etch electron microscopy

The detailed morphology of macrophages involved in the uptake and intracellular processing of low density lipoprotein (LDL) and, ultimately, formation of macrophage-derived foam cells of atherosclerotic lesions has long fascinated investigators. This study examined localization of LDL in subcellular compartments of macrophage-derived intimal foam cells in cardiac valves isolated from rabbits by diet-induced hypercholesterolemia and, as an in vitro model of formation of foam cells, in cultured human monocyte-macrophages incubated for 2;-120 h with aggregated LDL produced by vortexing or phospholipase C lipolysis. The quasi-three-dimensional morphology of macrophages involved in endocytosis was preserved by ultrarapid freezing and freeze-etch microscopy in conjunction with thin-section electron microscopy. This approach produced unique images of subcellular compartments in human monocyte-macrophages involved in the uptake and processing of aggregated LDL with a clarity not previously reported. Three-dimensional ultrastructural analyses revealed a complex network of coated and uncoated vesicles, surface-connected saclike compartments, and endosomal/lysosomal compartments including the labyrinth of vesicular/tubular lysosomes all enmeshed in the microtubular, microfilament cytoskeletal network. These dynamic views of subcellular structures at the high resolution of the electron microscope provide an additional framework to better understand how lipoprotein particles are transported into, and processed within, macrophages during foam cell formation in atherogenesis.     

Tendon xanthomas in familial hypercholesterolemia are associated with a differential inflammatory response of macrophages to oxidized LDL

Tendon xanthomas (TX) are pathognomonic lipid deposits commonly found in familial hypercholesterolemia (FH) patients. The aim of this study was to determine whether macrophages from FH patients with TX (TX+) have higher predisposition to foam cells formation after oxidized LDL (oxLDL) overload than those from FH patients without TX (TX-), and if their differential gene expression profile could explain these different phenotypes. Total RNA pools from macrophages from FH patients TX+ and TX- were analyzed using Affymetrix oligonucleotide arrays to evaluate the gene expression profile in presence and absence of oxLDL. Also, the intracellular lipid content was measured by fluorescence flow cytometry. Results of these studies suggest that macrophages from FH subjects TX+ compared to those TX- have a differential response to oxLDL, since they show higher intracellular cholesterol ester accumulation and a differential gene expression profile. The gene array data were validated by relative quantitative real-time RT-PCR and quantitative ELISA in culture media and plasma samples. FH subjects TX+ showed increased plasma tryptase, TNF-alpha, IL-8 and IL-6 concentrations. We propose that TX formation are associated with higher intracellular lipid content, and higher inflammatory response of macrophages in response to oxLDL.     

Modified methylenedioxyphenol analogs lower LDL cholesterol through induction of LDL receptor expression

Although statin therapy is a cornerstone of current low density lipoprotein (LDL)-lowering strategies, there is a need for additional therapies to incrementally lower plasma LDL cholesterol. In this study, we investigated the effect of several methylenedioxyphenol derivatives in regulating LDL cholesterol through induction of LDL receptor (LDLR). INV-403, a modified methylenedioxyphenol derivative, increased LDLR mRNA and protein expression in HepG2 cells in a dose- and time-dependent fashion. These effects were apparent even under conditions of HMG-CoA reductase inhibition. Electrophoresis migration shift assays demonstrated that INV-403 activates SREBP2 but not SREBP1c, with immunoblot analysis showing an increased expression of the mature form of SREBP2. Knockdown of SREBP2 reduced the effect of INV-403 on LDLR expression. The activation of SREBP2 by INV-403 is partly mediated by Akt/GSK3β pathways through inhibition of phosphorylation-dependent degradation by ubiquitin-proteosome pathway. Treatment of C57Bl/6j mice with INV-403 for two weeks increased hepatic SREBP2 levels (mature form) and upregulated LDLR with concomitant lowering of plasma LDL levels. Transient expression of a LDLR promoter-reporter construct, a SRE-mutant LDLR promoter construct, and a SRE-only construct in HepG2 cells revealed an effect predominantly through a SRE-dependent mechanism. INV-403 lowered plasma LDL cholesterol levels through LDLR upregulation. These results indicate a role for small molecule approaches other than statins for lowering LDL cholesterol.     

Concanavalin A and the in vitro induction in macrophages of vacuolation and lysosomal enzyme synthesis

Concanavalin A (ConA) induced extensive vacuolation in mouse peritoneal macrophages. Electron microscopic observations on thin sections reveal that the vacuoles are essentially empty except for minute vesicles attached to their inner periphery. The vacuoles consist of irregular structures and are heterogeneous in size distribution. ConA-induced vacuoles exhibit high acid phosphatase activity, suggesting fusion between vacuoles and lysosomes. Induction of acid phosphatase in ConA-treated macrophages was studied under several cultivation conditions. ConA-treated macrophage cultures responded in increase in acid phosphatase activity early after exposure to the lectin, a significant increase recorded already after 1 h. When cultivated in 1% serum medium for 48 h, ConA-treated macrophages exhibit twice the activity of acid phosphatase at zero time as well as that of non-treated control cultures. The effect of ConA on thioglycolate-stimulated mouse peritoneal macrophages was also studied. Vacuole formation resulting from lectin binding and internalization is discussed in terms of possible lectin effects on membrane fluidity, fusion capacity, surface to volume conservation during vacuole formation, fusion of vacuoles with lysosomes and intravacuolar lysosomal enzyme activities. The phenomenon of lysosomal enzyme induction as a result of ConA treatment is being correlated with enzyme induction due to other stimuli.     

Plasmodium sporozoite interactions with macrophages in vitro: a videomicroscopic analysis

Studies of in vitro interactions between Plasmodium berghei sporozoites and peritoneal macrophages from mice and rats were performed. A videomicroscopic analysis was made of interactions observed by phase-contrast microscopy. Our results showed a diversity of dynamic interactions between sporozoites and macrophages that included no interaction, surface interaction without sporozoite interiorization, active sporozoite penetration, active penetration with subsequent sporozoite escape, macrophage destruction, and the formation of "tethers" or web-like structures by sporozoites that had actively invaded macrophages. Sporozoites are thus clearly capable of actively invading host macrophages and are not restricted to being phagocytosed for interiorization. The formation of "tethers" by the moving sporozoite might function in vivo by anchoring the sporozoite to the cells lining the lumen of the liver sinusoid. Active sporozoite motility appears to be a functional phenomenon involved in sporozoite invasion of host liver cells.     

The interaction of human apoB-containing lipoproteins with mouse peritoneal macrophages: a comparison of Lp(a) with LDL

Cholesteryl ester accumulation in macrophages and foam cell formation is believed to play an important role in atherogenesis. The effect of Lp(a) on the incorporation of [14C]oleate into cholesteryl esters was studied in mouse peritoneal macrophages. In view of the physico-chemical similarities between Lp(a) and LDL, the results were compared with those obtained with LDL. Native Lp(a) and LDL did not stimulate cholesteryl ester formation. Incubation of macrophages with Lp(a)- or LDL-dextran sulfate complexes caused a significant increase in cholesteryl ester formation. A similar effect was observed when Lp(a) or LDL were incubated with macrophages in the presence of antibodies directed against the specific Lp(a) apoprotein or against LpB. Treatment of Lp(a) with acetic anhydride or malondialdehyde (MDA) was followed by precipitation of most of the lipoprotein. Therefore, these modifications were not suitable to study the uptake of modified Lp(a) by macrophages. Studies with acetyl-LDL or MDA-treated LDL caused the well-known stimulation of [14C]oleate incorporation into cholesteryl esters. Thus, the modification of Lp(a) by sulfated polysaccharides or by treatment with antibodies yields similar cholesteryl ester deposition in mouse peritoneal macrophages as observed with modified LDL. This might be one mechanism by which Lp(a) exerts its atherogenicity.     

Molecular characteristics in Japanese patients with lipidosis: Novel mutations in metachromatic leukodystrophy and Gaucher disease

The characterization of mutations in Japanese patients with lipidosis, particularly in metachromatic leukodystrophy (MLD) and Gaucher disease has been studied in detail. Metachromatic leukodystrophy is characterized by an accumulation of sulfatide in nervous tissues and kidney due to a deficiency of arylsulfatase A (ASA). We analyzed the presence of three known mutant arylsulfatase A alleles in Japanese patients with MLD. Among 10 patients of Japanese patients with MLD, we found that allele 445A mutation has moderately high incidence and also homozygosity of this mutation results in the late infantile form. Allele 2381T was not found in Japanese patients. Furthermore, we found novel mutation which is G- to A mutation at the 1070 nucleotide of the ASA gene (designated 1070 A) in Japanese patients with juvenile onset. This mutation results in a amino acid substitution of Gly245 by Arg and found in heterozygote form. Our studies of molecular analysis in 10 Japanese patients with MLD indicate that Japanese MLD patients have unique characteristics of ASA mutations compared with those of Caucasian patients. On the other hand, Gaucher disease is the most prevalent sphingolipidosis, characterized by an accumulation of glucocerebroside in macrophage derived cells due to a deficiency of lysosomal hydrolase glucocerebrosidase. To study the molecular basis of Gaucher disease in Japanese patients, we analyzed the presence of the two known mutations (6433C and 3548A) in the glucocerebrosidase gene of 15 patients with Gaucher disease. We found that the 6433C and 3548A mutations occur in all subtypes of Japanese patients with Gaucher disease. Most frequent mutations among them was the 6433C mutation, 40% of 30 chromosomes, whereas the novel mutation of the 3548A found in Japanese patients with neuronopathic Gaucher disease was found in 20% (6 out of 30 chromosomes). The characteristics of these mutations in Japanese patients with Gaucher disease is different from those of Caucasian populations reported previously.     

Effects of oxidatively modified LDL on cholesterol esterification in cultured macrophages

Oxidative modification of low density lipoproteins (LDL) has been shown to cause accelerated degradation of LDL via the scavenger receptor pathway in cultured macrophages, and it has been proposed that this process might lead to cholesterol accumulation in macrophages in the arterial wall in vivo. However, oxidation of LDL is accompanied by a substantial reduction in LDL total cholesterol content and hence the amount of cholesterol delivered by oxidatively modified LDL may be less than that delivered by scavenger receptor ligands such as acetyl LDL which results in massive cholesterol accumulation in cultured macrophages. The present studies were done to determine whether the decrease in total cholesterol content during LDL oxidation was due to oxidation of cholesterol and cholesteryl ester, and to determine whether the resulting oxidized sterols could affect cholesterol esterification in cultured macrophages. It was found that when LDL prelabeled with [3H]cholesteryl linoleate was oxidized, there was a decrease in cholesterol mass but no change in radioactivity. The radioactive substances derived from cholesteryl linoleate appeared more polar than the parent compound when analyzed by reverse-phase liquid chromatography, but were not identical with free cholesterol. Thin-layer chromatography of oxidized LDL lipids confirmed the loss of esterified cholesterol, and revealed multiple new bands, some of which matched reference oxysterols including 7-ketocholesterol, 5,6-epoxycholesterol, and 7-hydroxycholesterol. In addition to oxysterols, oxidized cholesteryl esters were also present. Quantitation by gas chromatography indicated that 7-ketocholesterol was the major oxysterol present.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multinucleation of macrophages with water-solubilized lignin derivatives in vitro

When macrophages derived from bone marrow were incubated with lignin derivative at 10 micrograms/ml, they formed multinucleated giant cells containing more than sixty nuclei. Cine-microscopic observation revealed that the formation of the giant cells was due to cell fusion. This phenomenon was specific for primary-cultured macrophages, and was not observed with either a macrophage cell line or established cell lines derived from other tissues.     

Boundary layer considerations in a multi-layer model for LDL accumulation

Abstract

Boundary layer effects for Low-Density Lipoprotein (LDL) concentration problems in a multi-layer artery model are analyzed in this work. Both a straight artery and aorta-iliac bifurcation are analyzed. Mass, momentum and species governing equations are based on the porous media theory and solved with the commercial finite-element based code COMSOL Multiphysics. For the straight artery, various inlet velocities, arterial sizes and intramural pressure values are investigated. Results are presented in terms of concentration profiles close to the lumen/endothelium interface and boundary layer thickness. It is shown that the boundary layer is affected by all of the three analyzed parameters. The results in this work will further clarify the concentration polarization effects imposed by the arterial wall.     

Selective induction of delayed hypersensitivity T-effector and T-suppressor lymphocytes in vitro by haptenized bone marrow-derived macrophages

The role of various subpopulations of antigen-presenting macrophages in the induction of T-lymphocyte subpopulations has been difficult to study in the past. We have used an in vitro system of bone marrow cell culture both to induce T-effector (TDH) and T-suppressor (Ts) cells active in delayed-type hypersensitivity. Bone marrow-derived macrophages (BM-MA) grown in Teflon bag cultures were allowed to attach to culture dishes and were pulse-labeled with 2,4-dinitrobenzene sulfonate (DNBSO3). Spleen cell lymphocytes from nonsensitized BALB/c mice were cocultured with antigen-pulsed or control BM-MA for 3 days. The lymphocytes were harvested, and injected iv into BALB/c mice which were challenged within 1 hr after injection by painting the right ear with 2,4-dinitrofluorobenzene (DNFB, effector test) or sensitized with DNFB on 2 days following iv injection of the cells and challenged 5 days later (suppressor test). Ear swelling was measured 24 hr later to assess the effector or suppressor function of the in vitro educated lymphocytes. BM-MA grown for 5 days (BM-MA 5) in L-cell conditioned medium induced only TDH cells (Thy 1+, Lyt 1+2-) whereas BM-MA grown for 10 days in conditioned medium induced only Ts cells (Thy 1+, Lyt 1-2+). In both cases, induced TDH and Ts cells were antigen specific. Functionally, induced Ts cells suppressed the afferent limb of the delayed response. When DNP-BM-MA 5 and DNP-BM-MA 10 were used to induce TDH or Ts cells in vivo by subcutaneous or intravenous injection respectively, only BM-MA 5 were able to sensitize recipient mice. Both 5- and 10-day macrophage populations induced Ts cells in vivo. Functionally, these Ts cells appeared to act on the efferent limb of the delayed reaction. We conclude that different populations of antigen-presenting macrophages can preferentially induce TDH or Ts cells, perhaps depending on antigen presentation in association with class II antigens or on the functional state of the antigen-presenting cell.