Incubation Of Mouse Peritoneal Macrophages Research Articles

Apolipoprotein E-Deficient Lipoproteins Induce Foam Cell Formation by Activation of PERK-EIF-2α Signaling Cascade

Transformation of macrophages into foam cells by apolipoprotein (Apo) E-deficient, ApoB48-containing (E(-)/B48) lipoproteins has been shown to be associated with increased phosphorylation of eukaryotic initiation factor-2α (eIF-2α). The present report examined the causal relationship between eIF-2α phosphorylation and lipid accumulation in macrophages induced by E(-)/B48 lipoproteins. E(-)/B48 lipoproteins increased eIF-2α phosphorylation and cholesterol ester accumulation, while lipoprotein degradation decreased and lysosomal acid lipase and cathepsin B mRNA translation was inhibited in mouse peritoneal macrophages (MPMs). These responses were overcome by overexpression of a nonphosphorylatable eIF-2α mutant in MPMs. Incubation of MPMs with E(-)/B48 lipoproteins also increased the phosphorylation of RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK), but not other eIF-2α kinases. Overexpression of a nonphosphorylatable PERK mutant inhibited PERK and eIF-2α phosphorylation, and alleviated cholesterol ester accumulation induced by E(-)/B48 lipoproteins. PERK is an eIF-2α kinase activated by endoplasmic reticulum (ER) stress. Taken together, findings from this report suggest that induction of ER stress, i.e., activation of the PERK-eIF2α signaling cascade, is a mechanism by which E(-)/B48 lipoproteins down-regulate lysosomal hydrolase synthesis, inhibit lysosomal lipoprotein degradation, and increase intracellular lipoprotein and cholesterol ester accumulation, resulting in foam cell formation.

Mutation of eukaryotic initiation factor‐2 α at serine 51 inhibits foam cell formation

We previously reported that ApoE‐deficient, ApoB48‐containing (E?/B48) lipoprotein‐induced foam cell formation was associated with an increased phosphorylation of eukaryotic initiation factor‐2α (eIF‐2α), a hallmark feature of endoplasmic reticulum stress. The objective of this study is to examine the impact of a nonphosphorylatable eIF‐2α mutant on E?/B48 lipoprotein‐induced changes in gene expression and foam cell formation. We observed that incubation of mouse peritoneal macrophages (MPMs) with E?/B48 lipoproteins reduced the protein levels of lysosomal hydrolases and cholesterol transporters, and enhanced eIF‐2α phosphorylation. Transfection of MPMs with an eIF‐2α mutant, in which serine 51 was replaced by alanine, alleviated the suppressive effect of E?/B48 lipoproteins on lysosomal hydrolase and cholesterol transporter expression, increased macrophage degradation of E?/B48 lipoproteins, enhanced cholesterol efflux, and suppressed foam cell formation. These observations suggest that induction of eIF‐2α phosphorylation is a mechanism by which ApoE‐deficient lipoproteins transform macrophages into foam cells. NIH grant support: K01HL076623 and RR003032 (H.Y.), R01ES014471 (Z.G.).

2-Aminopurine Inhibits Lipid Accumulation Induced by Apolipoprotein E-Deficient Lipoprotein in Macrophages: Potential Role of Eukaryotic Initiation Factor-2α Phosphorylation in Foam Cell Formation

We previously reported that apolipoprotein (Apo) E-deficient, ApoB48-containing (E^–/B48) lipoproteins inhibited expression of lysosomal hydrolase and transformed mouse peritoneal macrophages (MPMs) into foam cells. The present study examined the effect of 2-aminopurine (2-AP), an inhibitor of eukaryotic initiation factor (eIF)-2α phosphorylation, on E^–/B48 lipoprotein-induced changes in gene expression and foam cell formation. Our data demonstrated that E^–/B48 lipoproteins enhanced phosphorylation of eIF-2α in macrophages. Incubation of MPMs with E^–/B48 lipoproteins inhibited the translation efficiency of mRNAs encoding lysosomal acid lipase, cathepsin B, and cation-dependent mannose 6 phosphate receptor, with a parallel reduction in the level of these proteins. Addition of 2-AP to the culture media alleviated the suppressive effect of E^–/B48 lipoproteins on lysosomal hydrolase mRNA translation, increased macrophage degradation of E^–/B48 lipoproteins, and inhibited foam cell formation. Transfection of MPMs with a nonphosphorylatable eIF-2α mutant also attenuated the suppressive effect of E^–/B48 lipoproteins on expression of lysosomal acid lipase, associated with a reduced accumulation of cellular cholesterol esters. This is the first demonstration that ApoE-deficient lipoproteins inhibit lysosomal hydrolase synthesis and transform macrophages into foam cells through induction of eIF-2α phosphorylation.

Adverse functions of IL‐17A in experimental sepsis

IL-17A is a proinflammatory cytokine produced by a variety of cells. In the current study, we examined the role of IL-17A in sepsis induced in mice by cecal ligation and puncture (CLP). IL-17A levels, which rose time-dependently in plasma after CLP, were not affected in the absence of alphabeta T cells or neutrophils. In sharp contrast, gammadelta T cell-knockout or gammadelta T cell-depleted mice displayed baseline IL-17A plasma levels after CLP. Neutralization of IL-17A by two different antibodies improved sepsis (survival from approximately 10% to nearly 60%). Unexpectedly, antibody treatment was protective, even when administration of anti-IL-17A was delayed for up to 12 h after CLP. These protective effects of IL-17A blockade were associated with substantially reduced levels of bacteremia together with significant reductions of systemic proinflammatory cytokines and chemokines in plasma. In vitro incubation of mouse peritoneal macrophages with lipopolysaccharide (LPS) in the copresence of IL-17A substantially increased the production of TNF-alpha, IL-1beta, and IL-6 by these cells. These data suggest that, during experimental sepsis, gammadelta T cell-derived IL-17A promotes high levels of proinflammatory mediators and bacteremia, resulting in enhanced lethality. IL-17A may be a potential therapeutic target in sepsis.

Apolipoprotein E-deficient lipoproteins induce foam cell formation by downregulation of lysosomal hydrolases in macrophages

Apolipoprotein E (apoE) deficiency has been suggested to induce foam cell formation. Using lipoproteins obtained from wild-type mice and apoE-deficient mice expressing apoB-48 but not apoB-100, we studied apoE-deficient lipoprotein-induced changes in lipoprotein catabolism and protein expression in mouse peritoneal macrophages (MPMs). Our data demonstrate that incubation of MPMs with apoE-deficient lipoproteins induced intracellular lipoprotein, cholesteryl ester, and triglyceride accumulation, which was associated with a time-related decline in apoE-deficient lipoprotein degradation in MPMs. Confocal microscopy analysis indicated that the accumulated lipids were localized in lysosomes. ApoE-deficient lipoproteins reduced the protein levels of lysosomal acid lipase, cathepsin B, and cation-dependent mannose 6 phosphate receptor (MPR46). Exogenous apoE reduced apoE-deficient lipoprotein-induced lipid accumulation and attenuated the suppressive effect of apoE-deficient lipoproteins on lysosomal hydrolase and MPR46 expression. Although oxidized lipoproteins also increased lipid contents in MPMs, exogenous apoE could not attenuate oxidized lipoprotein-induced lipid accumulation. Our in vivo studies also showed that feeding apoE-deficient mice a high-fat diet resulted in cholesteryl ester and triglyceride accumulation and reduced lysosomal hydrolase expression in MPMs. These data suggest that apoE-deficient lipoproteins increase cellular lipid contents through pathways different from those activated by oxidized lipoproteins and that reducing lysosomal hydrolases in macrophages might be a mechanism by which apoE-deficient lipoproteins result in intralysosomal lipoprotein accumulation, thereby inducing foam cell formation.

The role of lipolysis in mediating the proinflammatory effects of very low density lipoproteins in mouse peritoneal macrophages

Hypertriglyceridemia is an important risk factor for atherosclerosis, especially in obesity. Macrophages are one of the primary cell types involved in atherogenesis and are thought to contribute to lesion formation through both lipid accumulation and proinflammatory gene expression. In this study, we sought to determine the direct impact of triglyceride (TG)-rich VLDL-induced lipid accumulation on macrophage proinflammatory processes. Incubation of mouse peritoneal macrophages with 100 microg/ml VLDL for 6 h led to 2.8- and 3.7-fold increases in intracellular TGs and FFAs, respectively (P < 0.05). The inflammatory proteins tumor necrosis factor-alpha, interleukin-1beta, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, matrix metalloproteinase 3 (MMP3), and macrophage inflammatory protein-1alpha (MIP-1alpha) were all upregulated by at least 2-fold (P < 0.05) in a dose-dependent manner in VLDL-treated macrophages. The increase in inflammatory gene expression coincided with the phosphorylation of the mitogen-activated protein kinase (MAPK) pathway members extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38 MAPK and was ameliorated by U0126, an inhibitor of ERK1/2. Inhibition of extracellular TG hydrolysis with tetrahydrolipstatin (Orlistat) resulted in the absence of intracellular TG and FFA accumulation and was accompanied by the amelioration of ERK1/2 phosphorylation and MIP-1alpha gene expression. These data indicate that VLDL hydrolysis, and the subsequent accumulation of intracellular FFAs and TGs, plays a substantive role in mediating the proinflammatory effects of VLDL. These data have important implications for the direct proatherogenic effects of VLDL on macrophage-driven atherosclerosis.

Activation of p38 MAPK is required for ligand‐induced Class A scavenger receptor expression

Class A Scavenger Receptors (SR-A) mediate the internalization of a variety of polyanionic proteins (e.g., modified LDL, bacteria) and adhesion to modified extracellular matrix. Previous studies demonstrated that SR-A expression and function are regulated by SR-A ligands. In this study, we determined the importance of specific intracellular signaling pathways in ligand-dependent regulation of SR-A expression. For this, we incubated mouse peritoneal macrophages (MPM) with the SR-A specific ligand AcLDL in the presence or absence of specific signaling pathway inhibitors. Activation of specific signaling proteins and changes in SR-A protein were assessed by western blotting cell lysates with antibodies to SR-A or the activated-state of specific signaling proteins. We found that incubating MPM with acetylated-LDL (AcLDL), but not native LDL, for 24 hrs dose-dependently increased SR-A expression. Incubation of MPM with AcLDL was associated with a rapid activation of the mitogen activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK1/2), cJun N-terminal kinase (JNK), and p38 kinase. To determine the importance of MAPK activation in ligand-dependent regulation of SR-A expression, MPM were treated with specific inhibitors of ERK1/2, JNK, and p38 prior to incubating with AcLDL. Ligand-induced SR-A expression was not affected by either ERK1/2 or JNK inhibitors but was abolished by pretreating cells with the p38 kinase inhibitor. Overall, these results indicate that ligand binding to SR-A activates multiple intracellular signaling pathways. However, ligand-induced SR-A expression specifically requires activation of p38 MAPK. (Supported by grants from the NIH and AHA)

Synergism between platelet-activating factor-like phospholipids and peroxisome proliferator-activated receptor gamma agonists generated during low density lipoprotein oxidation that induces lipid body formation in leukocytes.

Oxidized low density lipoprotein (LDL) has an important proinflammatory role in atherogenesis. In this study, we investigated the ability of oxidized LDL (oxLDL) and its phospholipid components to induce lipid body formation in leukocytes. Incubation of mouse peritoneal macrophages with oxidized, but not with native LDL led to lipid body formation within 1 h. This was blocked by platelet-activating factor (PAF) receptor antagonists or by preincubation of oxLDL with rPAF acetylhydrolase. HPLC fractions of phospholipids purified from oxLDL induced calcium flux in neutrophils as well as lipid body formation in macrophages. Injection of the bioactive phospholipid fractions or butanoyl and butenoyl PAF, a phospholipid previously shown to be present in oxLDL, into the pleural cavity of mice induced lipid body formation in leukocytes recovered after 3 h. The 5-lipoxygenase and cyclooxygenase-2 colocalized within lipid bodies formed after stimulation with oxLDL, bioactive phospholipid fractions, or butanoyl and butenoyl PAF. Lipid body formation was inhibited by 5-lipoxygenase antagonists, but not by cyclooxygenase-2 inhibitors. Azelaoyl-phosphatidylcholine, a peroxisome proliferator-activated receptor-gamma agonist in oxLDL phospholipid fractions, induced formation of lipid bodies at late time points (6 h) and synergized with suboptimal concentrations of oxLDL. We conclude that lipid body formation is an important proinflammatory effect of oxLDL and that PAF-like phospholipids and peroxisome proliferator-activated receptor-gamma agonists generated during LDL oxidation are important mediators in this phenomenon.

Oxidative stress increases the expression of the angiotensin-II receptor type 1 in mouse peritoneal macrophages.

Angiotensin II (Ang II) has been shown to accelerate atherogenesis, and the cellular Ang II type 1 (AT(1))-receptor mediates most of Ang II-induced pro-atherogenic effects. In this study we have examined the effect of macrophage oxidative stress on cellular AT(1)-receptor expression. Mouse peritoneal macrophages (MPM) from apolipoprotein-E deficient (E(0)) mice at increasing ages (1 6 months) demonstrated an age-dependent increase in cellular lipid-peroxides (PD) content. In parallel, the AT(1)-receptor mRNA and protein levels both increased by up to 3.7-fold and 1.7-fold, respectively, in MPM from 6-month old mice compared with 1-month old mice. Vitamin E supplementation to E(0) mice significantly decreased the MPM PD content and macrophage AT(1)-receptor mRNA expression compared with placebo-treated mice. The role of oxidative stress in the cellular expression of AT(1)-receptors was further demonstrated by manipulation of macrophage glutathione content. Buthionine-sulfoximine, a glutathione synthesis inhibitor, increased MPM PD content and AT(1)-receptor mRNA expression, whereas L-2-oxothiazolidine-4-carboxylic acid, that contributes to glutathione synthesis, reduced macrophage PD and AT(1)-receptor mRNA expression. Incubation of MPM with oxidised low-density lipoproteins (LDL) led to a significant, dose-dependent and time-dependent increase in macrophage AT(1)-receptor mRNA and protein expression, compared with control cells. In contrast, native LDL or acetylated LDL did not significantly affect macrophage AT(1)-receptor mRNA expression. In conclusion, our findings suggest that oxidative stress in macrophages induces AT(1)-receptor expression. This phenomenon can stimulate the interaction of Ang II with macrophages and hence accelerate macrophage foam cell formation and early atherogenesis.

Inhibition of cholesteryl ester formation in macrophages by azole antimycotics

Cultured macrophages take up and metabolize cholesterol-containing liposomes, resulting in massive accumulation of cholesteryl esters in the cells. Using this system, the effects of azole antimycotics on cholesteryl ester formation were studied. Incubation of mouse peritoneal macrophages with ketoconazole, miconazole, or econazole (0.1–10 μM) resulted in concentration-dependent inhibition of cholesteryl ester synthesis from endocytosed cholesterol. ic 50 values (concentration resulting in 50% inhibition) were 1.4 ± 0.1 μM, 4.1 ± 0.2 μM, and 3.6 ± 0.2 μM for ketoconazole, miconazole, and econazole, respectively. Complete inhibition was observed with 10 μM ketoconazole, and miconazole and econazole, each at 10 μM, caused 70 and 75% inhibition, respectively, of cholesteryl ester synthesis. The mechanism underlying the inhibition by ketoconazole was further studied. Ketoconazole did not appreciably block the uptake of liposomes or formation of triacylglycerol up to 10 μM. Interestingly, ketoconazole suppressed only 30% of 25-hydroxycholesterol-induced endogenous cholesterol esterification under conditions where esterification of endocytosed cholesterol was completely inhibited. Cytochemical studies with filipin-cholesterol staining revealed that ketoconazole induced massive accumulation of endocytosed cholesterol in macrophage phagolysosomes. These results indicate that ketoconazole inhibits cholesteryl ester formation in macrophages by blocking the intracellular transport of endocytosed cholesterol from lysosomes to the endoplasmic reticulum.

Angiotensin II Increases Macrophage-mediated Modification of Low Density Lipoprotein via a Lipoxygenase-dependent Pathway

The molecular and cellular mechanisms by which hypertension enhances atherosclerosis are poorly understood. Angiotensin II (Ang II) has been implicated in the regulation of cellular lipoxygenases (LO), which are thought to play a role in atherogenesis by inducing oxidative modification of low density lipoprotein (LDL). We sought to test the hypothesis that Ang II would stimulate murine macrophage LO activity (which has both 12- and 15-LO activity). Competitive binding studies revealed the presence of Ang II AT1 receptors on mouse peritoneal macrophages (MPM) and J-774 cells, but not on the RAW cell line. Valsartan, a specific AT1 receptor antagonist inhibited Ang II binding, whereas PD 123319, an AT2 receptor antagonist did not. Incubation of MPM or J-774 cells with Ang II (10 pM to 1 microM) for 24 h led to a 2.5-3.5-fold increase in LO activity, measured as generated 13-HODE or 12(S)-HETE. This stimulation was inhibited by valsartan, but not by PD 123319. In contrast, Ang II did not stimulate LO activity in RAW macrophages. Semiquantitative reverse transcriptase-polymerase chain reaction showed a 2-3-fold increase in LO mRNA in MPM, but not in RAW cells after treatment with Ang II. Ang II also induced an increase in 12-LO protein. In addition, pretreatment of J-774 cells with Ang II increased in a dose-dependent manner the ability of the cells to modify LDL, resulting in greater chemotactic activity for monocytes, typical of minimally modified LDL. This stimulation was inhibited by AT1 receptor blockade. In summary, these data suggest that Ang II increases macrophage LO activity via AT1 receptor-mediated mechanisms and this further increases the ability of the cells to generate minimally oxidized LDL. These studies provide a link between hypertension and the associated increased atherosclerosis observed in hypertensive patients.

Structural definition of a potent macrophage activating factor derived from vitamin D 3-binding protein with adjuvant activity for antibody production

Incubation of human vitamin D 3-binding protein (Gc protein), with a mixture of immobilized β-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized β-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10–50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2–4 days.

Enhanced LDL oxidation by murine macrophage foam cells and their failure to secrete nitric oxide

Foam cells were produced in vitro by incubation of mouse peritoneal macrophages with acetylated or copper oxidized LDL. Nitric oxide synthesis was stimulated by exposure of the cells to IFNy and LPS. Nitric oxide production, detected by measurement of nitrite in the culture medium, was unchanged in Ac-LDL loaded cells as compared with non-loaded cells. However, Ox-LDL foam cells produced 68–99% less nitrite than non-loaded cells. Failure to detect nitric oxide synthase (NOS) products from macrophages previously loaded with Ox-LDL appeared to result from lack of NOS activity, as little active enzyme could be recovered from Ox-LDL loaded cells. However, addition of Ox-LDL to an active cell-free NOS preparation had no direct effect on enzymic activity. When native LDL was subsequently incubated with these various IFNy/LPS stimulated cells, cells pre-loaded with Ox-LDL promoted, on average, a 2-fold greater increase in oxidative modification of the LDL added than either non-loaded or Ac-LDL loaded cells. That is, there was an inverse correlation between NOS activity and the ability of the cells to promote LDL oxidation. Unstimulated Ox-LDL loaded foam cells also oxidized LDL better than unstimulated non-loaded or Ac-LDL loaded foam cells, and the extent of oxidative modification was generally greater than seen with the equivalent IFNγ/LPS stimulated cells. This suggests that Ox-LDL loading also affects some additional factor(s) responsible for cell-mediated LDL oxidation.

Interaction of reconstituted high density lipoprotein discs containing human apolipoprotein A-I (ApoA-I) variants with murine adipocytes and macrophages. Evidence for reduced cholesterol efflux promotion by apoA-I(Pro165–>Arg).

Interaction of cells with both native and reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I mediates efflux of cellular cholesterol. To characterize structural requirements for this activity in apoA-I, six different genetic apoA-I variants and the corresponding normal allele products were isolated from heterozygous carriers, reconstituted with dimyristoylphosphatidylcholine (DMPC) into discoidal HDL and compared with regard to their ability to release intracellular cholesterol from murine adipocytes and peritoneal macrophages. In previous studies we determined the amino acid changes of these variants: Pro3-->Arg, Pro4-->Arg, Lys107-->0, Lys107-->Met, Pro165-->Arg, and Glu198-->Lys (von Eckardstein, A., Funke, H., Walter, M., Altland, K., Benninghoven, A., and Assmann, G. (1990) J. Biol. Chem. 265, 8610-8617) and demonstrated that all apoA-I variants except apoA-I(Lys107-->0) form discoidal HDL particles with phosphatidylcholine analogues indistinguishable from normal apoA-I (Jonas, A., von Eckardstein, A., Kezdy, K. E., Steinmetz, A., and Assmann, G. (1991) J. Lipid Res. 32, 95-106). In the present study, all apoA-I.DMPC complexes except those containing apoA-I(Pro165-->Arg) promoted cholesterol efflux as effectively as those containing normal apoA-I. Cholesterol efflux from adipocytes obtained after 180 min of incubation with apoA-I(Pro165-->Arg).DMPC complexes was decreased by 30% although this variant was bound to adipocytes with normal affinity. By contrast, apoA-I(Lys107-->Met).DMPC complexes were decreased in their binding affinity compared to normal apoA-I.DMPC complexes but normally promoted cholesterol efflux. Incubation of mouse peritoneal macrophages with apoA-I(Pro165-->Arg).DMPC complexes did also result in a 30% decreased efflux of radiolabeled cholesterol if compared to rHDL with the normal allele product from the same donor. Together the observations suggest that the substitution of proline at residue 165 interferes with the formation of a structural domain in apoA-I that is crucial for cellular cholesterol efflux stimulation. Furthermore, our results suggest that binding of HDL to adipocytes and cholesterol efflux promotion by HDL requires different structural domains.

Platelet-Modified Low Density Lipoprotein Induces Macrophage Cholesterol Accumulation and Platelet Activation

Low density lipoprotein (LDL), modified by chemical or biological means, was shown to induce macrophage cholesterol accumulation. The cholesterol and protein contents of LDL were decreased (by 10 and 15%, respectively) by incubation of the LDL for 2 h at 37 degrees C with normal washed platelet suspension or with platelet-conditioned medium; these decreases were not affected by platelet activation. The platelet-modified LDL caused a greater increase (by up to 15%) in collagen-induced, in vitro platelet aggregation than control LDL. Incubation of mouse peritoneal macrophages with platelet-modified LDL for 18 h at 37 degrees C resulted in an elevation of the macrophage cholesterol ester content (by 35-50%) as well as an increase in the cholesterol esterification rate (by 40-70%), compared with the effect of control LDL. Macrophage cholesterol synthesis, however, was significantly decreased (by 40-50%), compared with the effect of control LDL. The effect of LDL treated by platelet-conditioned medium was similar to that of platelet-modified LDL. The effect of platelet-modified LDL on macrophage cholesterol esterification was maximal within 24 h of incubation, and it was not significantly affected by inhibition of cholesterol synthesis. The platelet-modified LDL was taken up by the macrophages in a saturable fashion and its uptake was competitively inhibited by LDL, but not by acetylated LDL. We conclude that platelet-modified LDL interacts with the LDL receptor and induces macrophage cholesterol accumulation. Since the modified lipoprotein induces in vitro foam cell formation and platelet activation, platelet-modified LDL could be considered to be pro-atherogenic.

Accumulation of cholesteryl esters in macrophages incubated with human lipoprotein-antibody autoimmune complex

The interaction of mouse peritoneal and human pericardial macrophages with lipoprotein (LP)-antibody (Ab) immune complex isolated from the serum of ischemic heart disease (IHD) patients has been studied. It is shown that the Ab of the autoimmune complex belongs to IgG class, and the antigen is the LP with d < 1.063 g/ml. Incubation of mouse peritoneal macrophages with such complex led to the activation of [ 14C]oleic acid incorporation into cholesteryl esters by 2.5–2.8-fold, in comparison with the experiments where macrophages were incubated with free apoprotein (apo) B-containing LP isolated from the same serum. Incubation of human pericardial macrophages with autologous LP-Ab immune complex led to the transformation of macrophages into foam cells. These data lead to the conclusion that formation of LP-Ab autoimmune complexes may play an important role in the formation of atherosclerotic lesions of the arteries.

Enhanced macrophage uptake of low density lipoprotein after self-aggregation.

Incubation of mouse peritoneal macrophages with native human low density lipoprotein (LDL) did not cause any significant storage of intracellular cholesteryl esters. However, when the LDL was subjected to brief (30-second) vortexing, it formed self-aggregates that were rapidly ingested and degraded by macrophages, converting them to cholesteryl ester-rich foam cells. Such aggregates were as potent as acetyl-LDL in stimulating cholesterol esterification in the macrophages. The degradation of LDL aggregates was strongly inhibited by cytochalasin B (85%), whereas degradation of native LDL was only weekly inhibited (23%), suggesting that uptake occurred by phagocytosis rather than pinocytosis. Several lines of evidence suggest that the phagocytic uptake depends, in part, upon the LDL receptor and not the acetyl-LDL receptor: 1) soluble, native LDL and beta-VLDL (but not acetyl-LDL) competed for uptake and degradation of LDL aggregates; 2) reductive methylation of LDL before vortexing reduced the effect of the aggregates on degradation and cholesterol esterification; 3) heparin, which inhibits binding of native LDL to its receptor, reduced the degradation of LDL aggregates. These studies show that self-aggregation of LDL markedly enhances its uptake by macrophages, probably by phagocytosis and at least, in part, via the LDL receptor. Aggregates of LDL in the artery wall--either self-aggregates or mixed aggregates including matrix components--may induce foam cell formation and favor the formation of the fatty streak.

Effect of plasma lipoproteins on cholesterol accumulation in macrophages: comparison of lipoproteins from normal and homozygous familial hypercholesterolemic subjects

Total cholesterol (TC) content of mouse peritoneal macrophages (MPM) increased when incubated with increasing concentrations of normal low density (N-LDL) or very low density (N-VLDL) lipoprotein. Incubation with increasing concentrations of normal high density lipoprotein (N-HDL) caused a decrement in cellular mass of TC in MPM. Incubation of MPM with serum from normal subjects as well as from subjects with homozygous familial hypercholesterolemia (HFH) resulted in a 25% increment in cellular mass of TC, due to an increment in both free cholesterol (FC) and cholesteryl ester (CE) fractions. Accumulation of TC in MPM, due mainly to elevation of CE, was observed when the macrophages were incubated in the presence of LDL or VLDL derived from either group of subjects. N-LDL caused a higher increment in cellular CE compared to HFH-LDL. However, the presence of HFH-VLDL in the medium caused elevation in the cellular TC and CE content to a higher level than did N-VLDL. The presence of N-HDL as well as of HFH-HDL in the medium resulted in a similar decrement in the cholesterol content of MPM. The decrement was expressed in both FC and CE fractions. The present study shows different abilities of normal and HFH plasma lipoproteins to cause cholesterol accumulation in MPM.

Very low density lipoproteins promote triglyceride accumulation in macrophages.

Incubation of mouse peritoneal macrophages with very low density lipoproteins (VLDL) from normal rats or rhesus monkeys markedly increased the levels of intracellular triglycerides by 10- to 56-fold and was accompanied by the production of oil red O positive vacuoles. The stimulation of triglyceride accumulation in macrophages was time- and concentration-dependent and was specific for VLDL. Three possible mechanisms for the VLDL-stimulated triglyceride accumulation in macrophages were explored: receptor-mediated uptake, action of lipoprotein lipase, and phagocytosis. Macrophage uptake and degradation of 125I-monkey and rat VLDL demonstrated saturable and nonsaturable components. Uptake of 125I-VLDL could be inhibited by unlabeled normal VLDL, although hyperlipemic VLDL was more effective. HDL did not compete to a significant extent. Heparin released lipoprotein lipase-like activity from peritoneal macrophages. Addition of heparin with VLDL resulted in a greater, more rapid elevation in intracellular triglycerides, which was partially inhibited by albumin. Free fatty acid and Intralipid also produced triglyceride accumulation in macrophages. The data showed that all three of the mechanisms examined could contribute to the metabolism of VLDL by macrophages and cause the production of triglyceride-rich cells with a "foamy" appearance, although the evidence suggested that the action of lipoprotein lipase was probably the most important in this process.

EFFECT OF INTERFERON PREPARATIONS ON THE UPTAKE OF NON‐OPSONIZED ESCHERICHIA COLI BY MOUSE PERITONEAL MACROPHAGES

Uptake of non-opsonized Escherichia coli by mouse peritoneal macrophages (MPM) is influenced by pre-treatment of MPM with homologous interferon preparations. Incubation of MPM with 10(2)-10(3) units of interferon per ml for 18 hours resulted in a 30-70 per cent increase in the phagocytosis rate as well as maximal phagocytic capacity. This effect was time dependent. At least seven hours of interferon pre-treatment and a phagocytosis period of 90 minutes were necessary for a statistically significant increase of the phagocytosis. Treatment of MPM with high concentrations of interferon (i.e. more than 10(4) units per ml) had a depressive effect on the phagocytosis. Both the enhancing and the depressive effects were characterized by the standard physico-chemical properties of interferons. Both effects were species specific. The phagocytosis enhancing effect was neutralized by anit-interferon serum.