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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3428-3432.)
© 1997 American Heart Association, Inc.

Articles

Evidence for Hormone-Sensitive Lipase mRNA Expression in Human Monocyte/Macrophages

Karen Reue; Robert D. Cohen; ; Michael C. Schotz

From the Lipid Research Laboratory, West Los Angeles VA Medical Center, and the Department of Medicine, University of California, Los Angeles, Calif.

Correspondence to Karen Reue, West Los Angeles VA Medical Center, Bldg 113, Room 312, 11301 Wilshire Blvd, Los Angeles, CA 90073. E-mail reuek{at}ucla.edu

	Abstract

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Abstract The role of hormone-sensitive lipase (HSL) in the hydrolysis of adipose tissue triacylglycerol to provide free fatty acids for energy requirements has been well established. However, the role of HSL in other tissues, including macrophages, is not well understood. The demonstration that HSL is capable of hydrolyzing cholesteryl esters at approximately the same rate as triacylglycerol raised the possibility that HSL activity in macrophages may influence the accumulation of cholesteryl esters in foam cells of atherosclerotic lesions. We and others have previously demonstrated that HSL mRNA is expressed in murine peritoneal macrophages and macrophage cell lines; however, it was previously reported that HSL mRNA is absent in human monocyte-derived macrophages, suggesting that a species difference may exist. To clarify this point, we have further examined the issue of HSL mRNA expression in human macrophages. In the current study, we demonstrate that HSL mRNA is detectable in human monocyte-derived macrophages and in the THP-1 human monocyte cell line using reverse transcription coupled to polymerase chain reaction (RT-PCR). Specific amplification of cDNA derived from mRNA was ensured by using primers that span an intron within the human HSL gene, and the identity of PCR products as HSL was confirmed by hybridization to HSL cDNA and by DNA sequencing. Using a semiquantitative PCR assay, we establish that HSL mRNA levels in monocyte/macrophages are approximately 1/40 the levels in human adipose tissue. These results indicate that further studies addressing the role of HSL in macrophage metabolism and its potential role in development of foam cells in human atherosclerotic lesions are warranted.

Key Words: lipases • foam cells • monocytes/macrophages • THP-1 macrophages

	Introduction

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Foam cells derived from circulating monocyte/macrophages are a hallmark of atherosclerotic lesions, detectable even at early stages of fatty streak lesion formation.^{1 2 3} Foam cells develop from the accumulation of excess cholesterol derived from the uptake of modified lipoproteins through unregulated cellular receptors, such as the scavenger receptor.⁴ The cholesterol is stored in the cytoplasm in the form of cholesteryl esters, which may undergo repeated hydrolysis and re-esterification or hydrolysis and release from the cell in the presence of appropriate cholesterol acceptors in the extracellular space, such as high density lipoprotein (reviewed in References 5 and 6^{5 6} ). Although it is well established that the enzyme acyl CoA:cholesterol acyl transferase is responsible for cholesterol esterification in macrophage foam cells, the identity of the neutral cholesteryl ester hydrolase responsible for the release of cholesterol is still uncertain.

Several lines of evidence suggest that hormone-sensitive lipase (HSL) accounts for part or all of the neutral cholesteryl ester hydrolase activity in macrophages. A signature feature of HSL is its activation via phosphorylation by cAMP-dependent protein kinase.⁷ The neutral cholesteryl ester hydrolase present in J774 and P388D₁ mouse macrophage cell lines is activated by cAMP-dependent protein kinase.^{8 9} Furthermore, cAMP protein kinase stimulates clearance of cholesteryl ester from J774 cells in the presence of HDL as a cholesterol acceptor.¹⁰ Additionally, Small and colleagues^{11 12} have shown that anti-HSL antibody completely inhibits the neutral cholesteryl ester hydrolase activity in mouse peritoneal macrophages and WEHI mouse macrophage cell line. Recently, this laboratory has reported that HSL activity in the J774.2 mouse macrophage cell line is diminished to 20% normal levels after sterol ester loading by incubation with 25-hydroxycholesterol.¹³ These results further support an association between HSL activity levels and cholesteryl ester accumulation in macrophages.

In addition to the evidence for HSL activity in macrophages, HSL mRNA has been detected in the J774 and P388D₁ mouse macrophage cell lines, as well as in mouse peritoneal macrophages, by using reverse transcriptase coupled to polymerase chain reaction (RT-PCR).^{14 15} However, the possibility that HSL macrophage expression might be peculiar to the mouse was raised by a report of failure to detect HSL mRNA in human monocyte-derived macrophages using the same technique.¹⁶ In contrast to the latter report, studies presented here clearly demonstrate that HSL mRNA is expressed in primary human monocyte/macrophages, as well as in the human THP-1 macrophage cell line. Using a semiquantitative PCR assay, we determined that HSL mRNA levels in macrophages are approximately 1/40 the levels in human adipose tissue. These results indicate that further studies addressing the role of HSL in macrophage metabolism and its potential role in development of foam cells in human atherosclerotic lesions are warranted.

	Methods

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Cell Culture
THP-1 cells were obtained from the American Type Culture Collection (ATCC TIB-202). Cells were grown to confluence in 100-mm dishes in RPMI 1640 medium (GIBCO-BRL) containing 10% fetal bovine serum, 2x10^-5 mol/L 2-mercaptoethanol, 100 U/mL penicillin, and 100 g/mL streptomycin. Human blood monocytes were obtained from a large pool of healthy donors by modification of the Recalde procedure.¹⁷ Monocyte/macrophages were suspended in 30% autologous serum in Iscove's modified Dulbecco's medium supplemented with 2 mmol/L glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL Fungizone. Monocyte suspensions were plated at 10⁷ cells per milliliter in 100-mm dishes and incubated for 7 days. Cells were washed in PBS and harvested by scraping for subsequent RNA isolation.

RNA Isolation
Total RNA was isolated from human adipose tissue that had been snap-frozen in liquid nitrogen and from cultured cells using TRIzol (GIBCO-BRL) as specified by the manufacturer, except that lipid was removed from the top of adipose tissue homogenates after the first centrifugation step.

RT-PCR
cDNA was synthesized from 10 µg total RNA using AMV reverse transcriptase and oligo dT primer (cDNA Cycle Kit, InVitrogen). PCR amplification was performed using one tenth of the resulting cDNA (or a dilution of the cDNA, as indicated in "Results") in a total volume of 50 µL containing 50 mmol/L KCl, 10 mmol/L Tris-HCl, pH 8.3, 2 mmol/L MgCl₂, 0.001% gelatin, 5% DMSO, 200 µmol/L dNTPs, and 0.01 µg/µL forward and reverse primers. All primer sequences are given in the Table. Three independent sets of HSL primers were used, including the primer set previously used by Contreras and coworkers,^{15 16} and two novel sets of primers for human HSL (referred to as HSL primer sets 1 and 2). Additional primers used were for human ß-actin, scavenger receptor, and monocyte chemoattractant protein (MCP)-1. Each pair of primers was designed to span an intron, such that products resulting from amplification of cDNA sequences could be distinguished from products that would arise from potential genomic DNA contamination. Analysis of primer secondary structure was carried out using PrimerSelect software (DNAStar, Inc), which provides free energy estimates for DNA-DNA interactions, including primer self dimers, primer-primer dimers, and hairpin configurations.

View this table: [in this window] [in a new window]   Table 1. Primers Used for PCR

PCR amplification was carried out in a PTC-100 thermal cycler (MJ Research) using a hot-start "touchdown" protocol in which Taq polymerase was added to samples after an initial denaturation step, and the annealing temperature was gradually decreased over a 10°C range.¹⁸ The initial reaction conditions were denaturation at 94°C for 1 minute, annealing at 65°C for 1 minute, and extension at 72°C for 2 minutes. The annealing temperature was decreased from 65°C by 0.5°C at each cycle for 20 cycles and maintained at 55°C for the final 10 to 20 cycles. For semiquantitative PCR experiments, products were removed at intervals (28, 32, 36, and 40 cycles).

Southern Blot Hybridization
PCR products (12 µL) were electrophoresed on 1% agarose and transferred to Hybond-N+ membrane (Amersham), UV cross-linked, and hybridized to radiolabeled mouse HSL cDNA (2x10⁶ cpm/mL) as described.¹⁴ Blots were exposed to phosphor screens for 12 to 48 hours and imaged on a Phosphorimager 451 using ImageQuant software (Molecular Dynamics).

Cloning of HSL cDNA From Human Macrophages and THP-1 Cells
PCR was performed with cDNA prepared from human macrophage and THP-1 cell cDNA using HSL primer set 1. The resulting 433-bp products were purified by extraction from agarose gel using Magic DNA Clean-up resin (Promega) and ligated into the pCR II TA cloning vector (Stratagene). Resulting plasmid clones were sequenced by using Sequenase Version 2.0 Sequencing Kit (Amersham) with T7 and SP6 primers, and analysis of sequence data was performed with LaserGene software (DNAStar, Inc).

	Results

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Detection of HSL mRNA in Human Monocyte/Macrophages and THP-1 Cells by RT-PCR
We and others have previously used RT-PCR to detect HSL mRNA in mouse monocyte/macrophages and macrophage cell lines. Using a single set of PCR primers with sequences conserved in both mouse and human HSL cDNA, Contreras et al^{15 16} reported that HSL mRNA is detectable in mouse, but not human, macrophages. However, in our hands, the PCR primer sequences used in those studies performed poorly, producing only a weak signal for HSL from human adipose, the tissue with most abundant HSL mRNA (data not shown). These observations led us to suspect that the reported inability to detect HSL mRNA in human macrophages may have been due to an unfortunate choice of primer sequences, rather than a true species difference between mouse and human. To test this hypothesis, we designed two additional sets of HSL primers (HSL primer sets 1 and 2; see Table) using computer software designed to optimize compatibility between the two primer sequences and minimize secondary structure interactions (self dimer, pair dimer, and hairpin formation) that are detrimental to specific amplification of target sequences. Both primer sets were designed such that the sequence for each primer in the pair occurs in a separate exon of the human HSL gene¹⁹ to avoid detection of genomic DNA that could potentially contaminate cellular RNA preparations.

We used the two HSL primer sets to examine HSL mRNA expression in human monocyte/macrophages and the human THP-1 monocyte cell line. THP-1 cells exhibit several typical monocyte/macrophage properties, including phagocytosis, production of lysozymes, and expression of Fc and C3b receptors.²⁰ cDNA was prepared from human adipose, THP-1 cells, and monocyte/macrophages obtained from two independent human donor pools. Amplification of cDNA with primers for ß-actin²¹ produced a 426-bp product from all cDNA samples that was visible with ethidium bromide stain after electrophoresis in agarose (Fig 1a, top). Amplification of the same cDNA samples for 30 cycles with HSL primer set 1 and analysis with ethidium bromide stain revealed the expected 433-bp band from adipose tissue and faint bands of the expected size from THP-1 cells and monocyte/macrophages, along with some additional nonspecific bands (not shown). To determine whether the 433-bp band in THP-1 and monocyte/macrophage samples represented HSL, PCR products were transferred to membrane and hybridized with HSL cDNA. A distinct HSL product was detected in both THP-1 cells and human monocyte/macrophages, indicating that HSL is expressed in these cells (Fig 1a, bottom). No product was detected when water was substituted for cDNA in the amplification reaction (Fig 1a, right lane) or when reverse transcriptase was omitted from the cDNA preparation (not shown). Identical results were obtained with HSL primer set 2, indicating that the detection of HSL is not peculiar to the primer set (data not shown). To confirm that the THP-1 cells and monocyte/macrophages used for HSL amplification expressed macrophage-specific mRNAs, PCR was performed with primers for the scavenger receptor²² and MCP-1.²³ As shown in Fig 1b, the cDNA preparations used for HSL amplification also gave positive results for these two markers, confirming their macrophage lineage.

View larger version (72K): [in this window] [in a new window]   Figure 1. Detection of HSL mRNA in human monocyte/macrophages and THP-1 cells by reverse-transcribed polymerase chain reaction (RT-PCR). a, RNA from human adipose tissue, THP-1 cells (two independent RNA preparations), and monocyte/macrophages (RNA preparations from two independent donor pools) was reverse transcribed and PCR amplified, using ß-actin primers or HSL primer set 1 (see Table). The 426-bp ß-actin product was produced in samples derived from adipose tissue, THP-1 cells, and monocyte/macrophages and visualized by ethidium bromide stain (indicated by upper arrow). The 433-bp HSL product was visualized in adipose tissue, THP-1 cells, and monocyte/macrophages after blotting and hybridization to radiolabeled HSL cDNA (indicated by lower arrow). PCR products were not detected in samples containing water instead of cDNA (right lanes). b, RT-PCR performed on THP-1 cell and monocyte/macrophage RNA samples with primers for scavenger receptor and monocyte chemoattractant protein-1 (MCP).

Human Macrophage HSL cDNA Identical to Adipose Tissue HSL cDNA Sequence
To definitively demonstrate that the PCR product from human macrophages was HSL, THP-1 cDNA was amplified with HSL primer set 1 for 40 cycles to increase the amount of product. The product from several reactions was pooled, purified from agarose gel, and subcloned into a plasmid vector. Individual colonies were isolated and sequenced using primers specific for plasmid sequences on both sides of the insertion site. Of eight clones analyzed, six had sequence identical to the published sequence for human HSL cDNA from adipose tissue (nts 2200 to 2633 in Reference 19¹⁹ ). Interestingly, the other two clones sequenced were identical to each other but distinct from HSL. The cloned fragment contained the entire sequence for both the forward and reverse HSL PCR primers used in the amplification but otherwise had little similarity to HSL sequence. Searches of nucleic acid and protein databases with this sequence revealed an exact match to a single sequence; that for an expressed sequence tag isolated from a normalized human infant brain library (GenBank accession number R13955). Aside from the nucleic acid sequence, no information is available about the function or expression of this sequence tag. It is most likely that this product arises from the hybridization of the PCR primers to a cDNA species not related to HSL but having fortuitous similarity to the HSL primer sequences. That the non-HSL product represents a very minor fraction of the amplification products produced with the HSL primers was confirmed by restriction digestion of noncloned PCR products, which exhibited the pattern expected for HSL (data not shown).

HSL mRNA Is Expressed in Human Monocyte/Macrophages at 3% of the Level in Adipose Tissue
To estimate the relative abundance of HSL mRNA in human macrophages compared with adipose tissue, we performed a semiquantitative PCR assay in which samples were removed at cycles 28, 32, 36, and 40 to monitor the appearance of product during the linear phase of amplification.²⁴ To allow direct comparison of HSL mRNA levels in adipose and macrophages, the adipose cDNA template was diluted over a 100-fold range to find the concentration at which PCR products from adipose and macrophages would appear at approximately the same PCR cycle. We found that the appearance of HSL product from undiluted THP-1 and monocyte cDNA occurred at a similar PCR cycle as product from adipose cDNA that had been diluted 40-fold, indicating that HSL mRNA is expressed in macrophages at approximately 1/40 (2% to 3%) the levels in adipose (Fig 2, bottom). As a control, cDNA samples from adipose, THP-1 cells, and monocyte/macrophages were diluted 30-fold and amplified with ß-actin primers (Fig 2, top).

View larger version (44K): [in this window] [in a new window]   Figure 2. Relative levels of HSL mRNA in human adipose tissue and monocyte/macrophages. Semiquantitative reverse-transcribed polymerase chain reaction (RT-PCR) analysis of HSL expression levels in adipose tissue, THP-1 cells, and monocyte/macrophages was performed by monitoring the appearance of amplification product at progressive intervals of the amplification reaction. PCR products were detected by ethidium bromide staining after agarose gel electrophoresis. As shown in the upper portion of the figure, all cDNA samples were diluted 1:30 for amplification with ß-actin primers, and PCR products were analyzed after 28, 32, 36, and 40 cycles. In the lower portion, adipose tissue cDNA was diluted 1:40, and THP-1 and monocyte/macrophage cDNA were used without dilution for amplification with HSL primer set 1.

	Discussion

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The studies presented here demonstrate that HSL mRNA is expressed in human monocyte/macrophages and in the human THP-1 macrophage cell line. HSL mRNA was detected using RT-PCR, and the identity of HSL product definitively established by hybridization to HSL cDNA and by direct DNA sequencing. The use of primers that span an intron in the HSL gene allows unambiguous assignment of the template, giving rise to the PCR product as cDNA, rather than genomic DNA. Furthermore, the macrophage character of cells expressing HSL mRNA was also established by detection of mRNA for scavenger receptor and MCP, two macrophage-specific proteins. Using a semiquantitative PCR assay, we estimated the levels of HSL mRNA in human monocyte/macrophages and THP-1 cells at approximately 1/40 (2% to 3%) the levels in human adipose tissue. This estimate is consistent with the 35- to 40-fold higher neutral cholesteryl ester hydrolase activity in mouse adipose compared with resident peritoneal macrophages and the mouse P388D₁ macrophage cell line.¹⁴

The detection of HSL mRNA in human macrophages is consistent with previous findings that HSL mRNA and activity are present in peritoneal macrophages and WEHI-3b, J774, and P388D₁ cell lines derived from mouse.^{8 11 12 14 15} The current results, however, disagree with a previous report that HSL mRNA is absent from human monocyte/macrophages.¹⁶ One possible explanation for the discrepancy between the present and previous findings is technical differences between the two studies in the choices of primers and conditions used for PCR. In initial studies, we utilized the primer sequences in Reference 16¹⁶ and achieved unsatisfactory amounts of HSL RT-PCR product from human adipose tissue, an abundant source of HSL mRNA. On analysis of those primer sequences, we found that they are predicted to form numerous stable secondary structures that may compromise their performance in PCR, including self dimer, primer dimer, and hairpin structures. Therefore, in designing additional HSL primers for this study, we chose primer sequences that avoid these features and the associated drawbacks for PCR. The HSL primer sets used here produced shorter amplification products than those reported previously, a feature that could also contribute to increased efficiency of amplification. We also made use of a temperature cycling protocol known as "touchdown" PCR, in which the initial annealing temperature is chosen to be higher than the expected annealing temperature for the primers and then decreased each cycle to a final touchdown temperature that is 10°C below the starting temperature.¹⁸ This strategy is designed to circumvent spurious priming, a problem that is compounded in cases in which target template is present at very low abundance, such as the case with HSL mRNA in macrophages. These modifications in the PCR conditions may have increased the sensitivity to allow detection of HSL mRNA present at low abundance in human macrophages.

The demonstration that HSL is expressed in human monocyte/macrophages suggests that this enzyme is responsible for some or all of the neutral cholesteryl ester hydrolase activity in these cells, as has been demonstrated for murine macrophages.¹¹ These results also establish the THP-1 cell line as a readily available tool for further evaluation of the role of HSL and other cholesteryl ester hydrolases in cholesterol metabolism and foam cell formation and for examining potential differences in cholesterol metabolism that have been proposed between mouse and human macrophages.¹⁶ Furthermore, the similar levels of HSL mRNA expression in murine and human macrophages indicates that results from transgenic and gene knock-out mouse models with altered levels of HSL expression in macrophages will have relevance to similar processes in humans. Both in the development and reversal of atherosclerotic lesions, it is the cholesteryl ester concentration in the lesion that is markedly altered. Thus, it is reasonable to speculate that HSL in macrophage foam cells may be an important factor in the turnover of this lipid component.

	Acknowledgments

 
This work was supported by the Veterans Administration and Glaxo-Wellcome. K. Reue is an Established Investigator of the American Heart Association. The authors thank Dr Brian J. Van Lenten for providing human monocyte/macrophages and PCR primers for MCP-1.

Received February 21, 1997; accepted April 15, 1997.

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