Polymerase chain reaction-based method for quantifying recruitment of monocytes to mouse atherosclerotic lesions in vivo: enhancement by tumor necrosis factor-alpha and interleukin-1 beta.

The critical role of monocyte recruitment in atherogenesis has been appreciated for some time. However, until recently, there have been no sufficiently sensitive methods for measuring rates of monocyte recruitment to the arterial wall in vivo. We have developed a novel highly sensitive method, based on the polymerase chain reaction, for quantitatively tracking DNA-marked monocytes and have adapted it for use in mice. We use the uniquely male gene, SRY:, on the Y chromosome as a gene marker. We transfuse monocytes from a male donor into a congenic female mouse, euthanize the mouse after 24 to 48 hours, and then quantify the arterial uptake of monocytes by quantitative polymerase chain reaction. This study describes the techniques used and their sensitivity and reproducibility and demonstrates the approach by assessing the effects of cytokines. In control low density lipoprotein receptor-negative mice, monocyte recruitment decreased slightly but significantly as lesions progressed. Intraperitoneal injection of a combination of tumor necrosis factor-alpha and interleukin-1 beta more than doubled the rate of monocyte recruitment into developing lesions. However, the response to the cytokines was much greater in younger mice with less advanced lesions than in older animals with more advanced lesions.

The death of Sir Thomas Stamford Raffles (1781-1826).

The authors reviewed a rare autopsy report of Sir Thomas Stamford Raffles and offer a fresh interpretation of the cause of his death, with illustrations on the implied findings.

A new approach to determining the rates of recruitment of circulating leukocytes into tissues: application to the measurement of leukocyte recruitment into atherosclerotic lesions.

Recruitment of circulating monocytes into the artery wall is an important feature of early atherogenesis. In vitro studies have identified a number of adhesion molecules and chemokines that may control this process but very little work has been done to evaluate their relative importance in vivo, in part because there have been no methods available of sufficient sensitivity and reliability. This paper proposes a new approach in which advantage is taken of naturally occurring or transgenically induced mutations to "mark" donor cells and to follow their fate in recipient animals using highly sensitive PCR methods. The feasibility of the approach is demonstrated by preliminary studies of monocyte recruitment into atherosclerotic lesions. However, the method should in principle be applicable to the study of any of the circulating leukocytes and their rate of entry into any tissue or tissues of interest.

Selective uptake of HDL cholesteryl esters is active in transgenic mice expressing human apolipoprotein A-I.

The direct non-endocytotic uptake of cholesteryl esters (CE) from high density lipoprotein (HDL) plays a major role in HDL CE metabolism in rats and rabbits. In vitro evidence indicates it may also play such a role in humans. However, a study in mice (tracing the CE and apoA-I moieties of HDL) concluded that, while selective uptake played a role in normal animals, it did not in transgenic mice which express predominantly human apoA-I (Chajek-Shaul et al., 1991. Proc. Natl. Acad. Sci. USA. 88: 6731-6735);thus human apoA-I was apparently unable to support selective uptake. These conclusions rested on plasma decay data that represent a composite of all tissue and which may obscure tissue-specific factors. Thus we reexamined the matter by measuring the rates of uptake of HDL components by individual tissues using intracellularly trapped tracers. Plasma decay data were much as reported in the referenced study. Nonetheless the fractional rate of uptake of HDL CE was greater than that of apoA-I in adrenal gland and liver, indicating selective uptake. Kidney took up apoA-I tracer at a greater fractional rate than CE tracer, apparently by filtration and reabsorption of free apoA-I, and this uptake was at a greater fractional rate in the transgenic mice than in normal mice. Thus, the lack of evidence for selective uptake in the plasma decay data of the transgenic mice was explained by a higher rate of renal uptake of apoA-I and not by a diminished rate of selective uptake in other tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of hormone-sensitive lipase mRNA in macrophages.

Macrophages contain a neutral cholesteryl ester hydrolase that can be activated by cAMP-dependent protein kinase. Immunological studies strongly suggest that hormone-sensitive lipase (HSL) is probably responsible for the cholesteryl ester hydrolase activity in macrophages; however, due to the very low level of expression in macrophages, it has been difficult to determine whether the macrophage cholesteryl ester hydrolase and adipose HSL are, in fact, products of the same gene. We have used the sensitive polymerase chain reaction (PCR) technique to demonstrate expression of HSL mRNA in resident and thioglycollate-elicited mouse peritoneal macrophages, as well as in the P388D1 mouse macrophage cell line. PCR was performed using oligonucleotide primer sequences present on adjacent exons of the mouse HSL gene to allow discrimination between products derived from HSL mRNA or genomic DNA sequences; specificity of the PCR was demonstrated by the absence of a product in liver, which does not express HSL mRNA. Northern blot analysis of poly (A)+ RNA from peritoneal macrophages with a mouse adipose HSL cDNA probe demonstrated a low abundance of mRNA of 3.2 kb, identical in size to HSL mRNA in adipose tissue. These findings, together with the results of previous studies demonstrating similarities between HSL and macrophage neutral cholesteryl ester hydrolase, strongly support the conclusion that both are products of a single gene. The development of a PCR assay for HSL mRNA may allow further study of the regulation of neutral cholesteryl ester hydrolase expression in macrophages and foam cells, and its potential role in atherogenesis.

Monoclonal antibodies against LDL further enhance macrophage uptake of LDL aggregates.

Self-aggregates of low density lipoprotein (LDL) are taken up and degraded more rapidly by macrophages than is native LDL. That enhanced uptake is attributable in part to phagocytosis via the LDL receptor pathway. However, arterial macrophages appear to express little LDL receptor activity. The present studies demonstrate an alternative mechanism by which LDL aggregates could contribute to foam cell formation. This could occur by the formation of large immune complexes that are taken up by macrophages via the Fc receptor. When immune complexes were formed with native, soluble LDL and MB47, a monoclonal antibody specific to the apoprotein B domain recognized by the LDL receptor, the subsequent uptake and degradation of the LDL by macrophages were inhibited 50-80% compared with native LDL alone. In contrast, when aggregated LDL was bound to MB47 at a similar molar ratio, the subsequent degradation of the insoluble immune complexes was two- to fivefold greater than that of aggregated LDL alone. The enhanced uptake was abolished when Fab or F(ab')2 fragments of MB47 were substituted for the intact antibody, indicating that the increased uptake was via the Fc receptor pathway. Furthermore, the uptake of the immune complexes of aggregated LDL was reduced by competition for the Fc receptor with heat-aggregated immunoglobulin. There was also an increase in the rate of cellular cholesterol esterification and an increase in macrophage cholesteryl ester mass. Since aggregates of LDL as well as autoantibodies against modified LDL have been demonstrated in atherosclerotic lesions, it is possible that immune complexes of aggregates of modified LDL may be generated in the intima.(ABSTRACT TRUNCATED AT 250 WORDS)

Feasibility of using an oleate-rich diet to reduce the susceptibility of low-density lipoprotein to oxidative modification in humans.

Oxidized low-density lipoprotein (LDL) is more atherogenic than native LDL. The initial step in the oxidation is the peroxidation of polyunsaturated fatty acids. Thus, decreasing the concentration of polyunsaturated fatty acids should reduce the susceptibility of LDL to oxidation. Therefore, we tested the possibility that diets enriched in oleate might result in LDL that is less susceptible to oxidative modification. LDL isolated from subjects consuming an oleate-enriched diet, compared with LDL from subjects on a linoleate-enriched diet, contained significantly more oleate (28.7% vs 11.5%) and less linoleate (31.9% vs 50.9%). Generation of conjugated dienes was significantly lower in the LDL from the oleate group. Most important, after incubation with endothelial cells, LDL from the oleate group underwent less degradation by macrophages. These studies demonstrate the feasibility of altering the diet in a way that will not raise LDL cholesterol concentrations and yet will decrease the susceptibility of LDL to oxidative modification.

Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation of low-density lipoproteins, and contain oxidation-specific lipid-protein adducts.

Pure macrophage-derived foam cells (MFC) were isolated from the aortas of rabbits made atherosclerotic by balloon deendothelialization followed by diet-induced hypercholesterolemia. The MFC were isolated under sterile conditions using an enzymatic digestion procedure and discontinuous density gradient centrifugation. The purity of the MFC preparations was verified immunocytochemically with the macrophage specific monoclonal antibody RAM-11. MFC plated in medium containing 0.5% FCS for 24 h contained approximately 600 micrograms cholesterol per mg cell protein, 80% of which was esterified cholesterol. The MFC specifically degraded low density lipoprotein (LDL), acetyl-LDL, copper oxidized LDL, and beta-very low density lipoprotein (beta-VLDL) at rates comparable to mouse peritoneal macrophages (MPM) in 5-h assays. MFC within sections of the atherosclerotic lesions from the ballooned rabbits as well as the MFC isolated from the same lesions in the presence of antioxidants, exhibited positive immunoreactivity with polyclonal guinea pig antisera and mouse monoclonal antibodies directed against malondialdehyde-LDL, and 4-hydroxynonal-LDL. The MFC also exhibited the capacity to induce the oxidation of LDL at rates comparable to those exhibited by MPM and rabbit aortic endothelial cells. These data provide direct evidence that arterial wall macrophages express modified LDL receptors in vivo, contain epitopes found in oxidized-LDL and are capable of oxidizing LDL even when maximally loaded with cholesterol.

Low density lipoprotein rich in oleic acid is protected against oxidative modification: implications for dietary prevention of atherosclerosis.

Oxidative modification of low density lipoprotein (LDL) enhances its potential atherogenicity in several ways, notably by enhancing its uptake into macrophages. In vivo studies in the rabbit show that inhibition of LDL oxidation slows the progression of atherosclerotic lesions. In the present studies, rabbits were fed either a newly developed variant sunflower oil (Trisun 80), containing more than 80% oleic acid and only 8% linoleic acid, or conventional sunflower oil, containing only 20% oleic acid and 67% linoleic acid. LDL isolated from the plasma of animals fed the variant sunflower oil was highly enriched in oleic acid and very low in linoleic acid. These oleate-rich LDL particles were remarkably resistant to oxidative modification. Even after 16-hr exposure to copper-induced oxidation or 24-hr incubation with cultured endothelial cells, macrophage uptake of the LDL was only marginally enhanced. The results suggest that diets sufficiently enriched in oleic acid, in addition to their LDL-lowering effect, may slow the progression of atherosclerosis by generating LDL that is highly resistant to oxidative modification.

Prevention of low density lipoprotein aggregation by high density lipoprotein or apolipoprotein A-I.

We have shown previously that low density lipoprotein (LDL) subjected to vortexing forms self-aggregates that are avidly phagocytosed by macrophages. That phagocytic uptake is mediated by the LDL receptor. We now show that LDL self-aggregation is strongly inhibited (80-95%) by the presence of high density lipoprotein (HDL) or apolipoprotein (apo) A-I. Another type of LDL aggregation, namely that induced by incubation of LDL with phospholipase C, was also markedly inhibited by HDL or apoA-I. The aggregation of LDL induced by vortexing was not inhibited by 2.5 M NaCl, and apoA-I was still able to block LDL aggregation at this high salt concentration, strongly suggesting hydrophobic interactions as the basis for the effect of apoA-I. The fact that apoA-I protected against LDL aggregation induced by two apparently quite different procedures suggests that the aggregation in these two cases has common features. We propose that these forms of LDL aggregation result from the exposure of hydrophobic domains normally masked in LDL and that the LDL-LDL association occurs when these domains interact. ApoA-I, because of its amphipathic character, is able to interact with the exposed hydrophobic domains of LDL and thus block the intermolecular interactions that cause aggregation.

Stimulation of a neutral cholesteryl ester hydrolase by cAMP system in P388D1 macrophages.

Cholesteryl ester laden foam cells in atherosclerotic lesions derive, in part, from macrophages. Mobilization of stored cholesteryl esters involves hydrolysis by a neutral cholesteryl ester hydrolase. Incubation of intact P388D1 macrophages with dibutyryl cAMP in the presence of 1-methyl-3-isobutylxanthine resulted in a dose-dependent increase in neutral cholesteryl ester hydrolase activity of up to 50% (ED50 = 0.1 mM). Incubation with prostaglandin E1 in the presence of 1-methyl-3-isobutylxanthine also increased neutral cholesterol ester hydrolase activity by about 50%. In cell-free preparation, cAMP-dependent protein kinase caused about a 2-fold activation of the neutral cholesteryl ester hydrolase. Activation was blocked by protein kinase inhibitor. These data suggest that the P388D1 macrophage may be a useful model for studying the hormonal regulation of cholesteryl ester mobilization in macrophage-derived foam cells.

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.

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.

Secretion of the lysosomal acid triacylglycerol hydrolase precursor by J774 macrophages.

J774, thioglycollate-elicited mouse peritoneal and BCG-induced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The J774 macrophages, a cell line deficient in the calcium-independent mannose 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH4Cl induced a 6-fold increase in TGase secretion by alveolar macrophages, while 50 mM NH4Cl induced a 12-fold increase in TGase secretion. NH4Cl had no effect on TGase secretion by J774 macrophages. The TGase secreted by J774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase 10-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose 6-phosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-independent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase.

A rapid and sensitive enzymatic-radiochemical assay for the determination of triglycerides.

An enzymatic-radiochemical method suitable for the determination of triglyceride levels of cells in culture is described. The method is based on the enzymatic hydrolysis of triglycerides to free fatty acids which then complex with 63Ni. The method is rapid, accurate, and inexpensive. The procedure extends the sensitivity of triglyceride measurement to as low as 0.25 nanomoles.

Regulation of lipoprotein lipase secretion by mouse peritoneal macrophages.

Resident mouse peritoneal macrophages in culture spontaneously secrete lipoprotein lipase. Secretion of lipoprotein lipase was 10-fold greater in thioglycollate-elicited and 6-fold greater in mineral oil-elicited macrophages. The increase in enzyme secretion was dependent on protein synthesis and glycosylation of the newly synthesized enzyme. The addition of 1 to 500 ng per ml of lipopolysaccharide to the culture medium resulted in a dose-dependent inhibition of lipoprotein lipase secretion. Thus, secretion of macrophage lipoprotein lipase is a regulated process, and may be increased as part of the response to inflammation. This may have important implications in the atherogenic process in the arterial wall.

Activation of myocardial neutral triglyceride lipase and neutral cholesterol esterase by cAMP-dependent protein kinase.

Lipolysis of intracellular triglycerides in the heart has been shown to be regulated by hormones. However, activation of myocardial triglyceride lipase in a cell-free system has not been directly demonstrated. In the present studies, initial attempts to demonstrate cAMP-dependent activation of triglyceride lipase using the 1,000 X g supernatant fraction (S1) of mouse heart homogenate were unsuccessful, presumably due to the masking effects of high levels of lipoprotein lipase activity even when assayed at pH 7.4 and in the absence of apolipoprotein C-II. Myocardial lipoprotein lipase in the 40,000 X g supernatant fraction was then removed by heparin-Sepharose affinity chromatography. The lipoprotein lipase-free fractions were shown to contain neutral triglyceride lipase and neutral cholesterol esterase of about equal activities. The triglyceride lipase and cholesterol esterase activities fell progressively during preincubation in the presence of 5 mM Mg2+. Additions of cAMP and ATP resulted in 40-70% activation of both triglyceride lipase and cholesterol esterase. The activation was blocked by protein kinase inhibitor and was restored by the addition of exogenous cAMP-dependent protein kinase. Since lipoprotein lipase has no activity toward cholesteryl oleate, activation of cholesterol esterase in untreated S1 was readily demonstrable. Both triglyceride lipase and cholesterol esterase activities were present in homogenates prepared from isolated rat heart myocytes. We conclude that the myocardium contains a hormone-sensitive lipase that is regulated in a fashion similar to that of the adipose tissue enzyme.

Mobilization of stored triglycerides from macrophages as free fatty acids.

Because many or most lipid-laden foam cells in atheromas and in xanthomas derive from macrophages, it is important to understand how they accumulate lipids and how they can divest themselves of lipids. The mobilization of stored triglycerides from macrophages was studied in cell cultures. Mouse resident peritoneal macrophages and J774 macrophages increased their triglyceride content six- to tenfold during a 24-hour incubation with free fatty acids complexed to albumin. Subsequent incubation in fresh medium containing free fatty acid-poor albumin was accompanied by a fall in cell triglyceride content (50% in 20 hours) and a corresponding increase in medium-free fatty acid. Release of free fatty acid was linear as a function of time, provided fresh medium was added hourly. When medium was not changed, release rates fell off rapidly, probably due to re-uptake of released free fatty acid. Chloroquine did not affect the rate of free fatty acid release. The results suggest that macrophages-foam cells can reduce their triglyceride stores via the action of a nonlysosomal (presumably cytoplasmic) neutral triglyceride lipase.

Neutral triglyceride lipase in macrophages.

High levels of neutral triglyceride lipase activity have been demonstrated in several types of macrophages (J774 cells, human monocyte/macrophages, rabbit alveolar macrophages, and resident mouse peritoneal macrophages). The pH optima ranged from 6.5 to 7.4 depending upon the buffer and the conditions of incubation. The addition of bovine serum albumin stimulated activity at low concentrations, as expected for a fatty acid-releasing reaction, but strongly inhibited at higher concentrations; maximal activity was observed in the presence of 0.625 mg/ml of bovine serum albumin. The enzyme was remarkably thermostable, showing no apparent loss of activity at 50 degrees C for as long as 6 hours. The lipase was inhibited 80% by 0.1 M NaCl. Assayed under optimal conditions, the specific activity of the neutral triglyceride lipase from J774 cells was more than 100-fold greater than the activity of lipoprotein lipase or neutral cholesterol esterase from those cells; this activity was 10-fold greater than the levels of hormone-sensitive lipase from 3T3-L1 adipocytes. This neutral triglyceride lipase may play an important role in the degradation and mobilization of cytosolic triglyceride in macrophage-derived foam cells.