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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:392-399.)
© 1995 American Heart Association, Inc.

Articles

Lipoprotein Lipase Synergizes With Interferon Gamma to Induce Macrophage Nitric Oxide Synthetase mRNA Expression and Nitric Oxide Production

Geneviève Renier; Anick Lambert

From the Department of Nutrition, Notre-Dame Hospital Research Center, Metabolic Unit, University of Montreal, Montreal, Canada.

Correspondence to Dr Geneviève Renier, Notre-Dame Hospital, Metabolic Unit, Pavillon Mailloux, 8th Floor, 1560 Sherbrooke St E, Montreal, Quebec, Canada H2L 4M1.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract Lipoprotein lipase (LPL) induces macrophage tumor necrosis factor- (TNF-) gene expression and protein secretion. Since TNF- can increase interferon gamma (IFN-)–dependent nitric oxide (NO) production, we studied whether LPL may synergize with IFN- for the induction of macrophage NO production. Although ineffective by itself, LPL in combination with IFN- increased L-arginine–dependent NO production in a dose-dependent manner. Preincubation of LPL with an anti-LPL neutralizing antibody totally suppressed this effect. Increased NO synthetase (NOS) mRNA expression was also observed after macrophage treatment with IFN- and LPL. Protein synthesis was required for the induction of NOS mRNA, and a TNF-–mediated effect of LPL on NOS gene expression and NO production was observed. The ability of LPL to augment IFN-–dependent NOS mRNA expression was associated with an increase in the NOS gene transcriptional activity but not in the NOS mRNA stability. Finally, binding of nuclear proteins to the nuclear factor–B– and TNF-–responsive sequences of the macrophage NOS promotor was decreased by treatment of the cells by IFN- alone or in combination with LPL. These data provide evidence for a link between LPL and arginine metabolism in macrophages and further stress the role of LPL in the regulation of macrophage activation.

Key Words: lipoprotein lipase • tumor necrosis factor– • interferon gamma • macrophage • nitric oxide

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Nitric oxide (NO) is a major effector molecule involved in the generation of tumoricidal and microbicidal activities by activated macrophages.¹ It is formed by macrophages as a product of the conversion of L-arginine to L-citrulline by NO synthetase (NOS). Activated macrophages express large amounts of the inducible, cytosolic calcium–independent NOS. Different stimuli, including interferon gamma (IFN-) alone or in combination with lipopolysaccharide (LPS), tumor necrosis factor– (TNF-), and interleukin-2 have been reported to induce macrophage NOS.^{2 3 4 5}

Lipoprotein lipase (LPL), a key enzyme in the metabolism of triglyceride-rich lipoproteins,⁶ is constitutively expressed by macrophages⁷ and synthesized by parenchymal cells of various tissues.^{8 9 10 11 12} Treatment of macrophages with LPL induces TNF- gene expression and secretion,¹³ suggesting an autocrine effect of LPL on macrophage activation. These results and the well-known synergistic effect of IFN- and TNF- on macrophage NO production led us to investigate the possibility that LPL may synergize with IFN- to increase macrophage NO formation.

The present study provides the first evidence that L-arginine–dependent NO production may be induced by LPL in combination with IFN- and that this effect is at least partly due to an increase in NOS gene transcriptional activity. In addition, it shows that TNF- acts, at least partly, as a mediator of the observed LPL effects. Overall, these data further suggest that LPL may be important for the activation and expression of effector functions by murine macrophages.

	Methods

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Reagents
Fetal bovine serum was purchased from Hyclone Laboratories. Dulbecco's minimal essential medium (DMEM) was obtained from ICN Biochemicals Inc and supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine (ICN Biochemicals Inc), and penicillin-streptomycin (Flow). Cycloheximide (CHX) and polymyxin B sulfate were purchased from Sigma Chemical Co. Recombinant murine TNF- and polyclonal anti-mouse TNF- were obtained from Genzyme. Purified anti-bovine LPL antibody was a generous gift from Dr Juan DeSanctis (Institute of Immunology, Caracas, Venezuela). Recombinant murine IFN- was purchased from GIBCO BRL.

Purification of LPL
LPL was isolated from human postheparin plasma. The enzyme was purified¹⁴ by using two column steps of heparin-Sepharose affinity chromatography and elution with 2 mol/L NaCl. Protein purity was tested by Western blot analysis.¹⁵

In some experiments, polymyxin B (100 µg/mL) was added to the LPL preparations. This treatment did not modify the effect of LPL on NO production.

Determination of Endotoxin Concentrations
The endotoxin content of all LPL preparations was determined by a quantitative Limulus amoebocyte lysate assay (Whittaker). The endotoxin content in the LPL preparations was consistently found to be lower than 6 pg/mL.

Macrophages
We used the ANA-1 macrophage line established by infection of the normal bone marrow of C57BL/6 mice with the J2 recombinant retrovirus.¹⁶ ANA-1 cells have been characterized as a homogeneous population of macrophages on the basis of their characteristic morphology, lack of T- or B-cell markers, and cell surface expression of specific markers or antigens expressed strongly by macrophages. They did not constitutively express major histocompatibility complex class II I-A region antigens nor exhibit constitutive tumoricidal activity, indicating that they are not activated macrophages. The macrophage line was cultured in DMEM containing 10% fetal bovine serum and treated for different time periods with the appropriate agents.

RNA Extraction
Ten million macrophages were plated in plastic Petri dishes (Falcon). Following the treatment of macrophages with activating agents, macrophages were lysed with guanidine isothiocyanate. Total RNA was purified by centrifugation through a cesium chloride gradient as detailed by Chirgwin et al.¹⁷

Northern Blot Analysis
Total RNA (15 µg) was separated on a 1.2% agarose gel containing 2.2 mol/L formaldehyde.¹⁸ The blots were prehybridized for 18 hours in prehybridization buffer. The mRNA expression was analyzed by hybridization with [-³²P]dCTP (specific activity 3000 Ci/mmol; Amersham Corp)–labeled NOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA inserts. Hybridization was detected by autoradiography with Kodak X-Omat-AR films. RNA expression was quantified by high-resolution optical densitometry (SciScan 5000, USB).

Nuclear Run-on Assay
Nuclear run-on experiments were performed as described by Greenberg and Ziff.¹⁹ [-³²P]UTP-labeled RNA (2x10⁶ cpm/mL) was hybridized to NOS, GAPDH, and pBluescript DNA linearized probes spotted to nitrocellulose.

DNA Binding Assays
The isolation of nuclei was performed as described.²⁰ Briefly, 5x10⁷ ANA-1 cells were collected, washed with cold phosphate-buffered saline, and lysed in 1 mL ice-cold buffer A (15 mmol/L KCl, 2 mmol/L MgCl₂, 10 mmol/L HEPES, 0.1 mmol/L EDTA, 1 mmol/L dithiothreitol [DTT], 2 µg/mL aprotinin, 0.1% phenylmethylsulfonyl fluoride [PMSF], and 0.5 % Nonidet P-40). After a 10-minute incubation on ice, lysed cells were centrifuged, and the nuclei were washed with buffer A without Nonidet P-40. The nuclei were then lysed in a buffer containing 2 mol/L KCl, 25 mmol/L HEPES, 0.1 mmol/L EDTA, and 1 mmol/L DTT. After a 15-minute incubation period, a dialysis buffer (25 mmol/L HEPES, 1 mmol/L DTT, 0.1% PMSF, 2 µg/mL aprotinin, 0.1 mmol/L EDTA, and 11% glycerol) was added to the nuclei preparation. Following centrifugation for 20 minutes at 13 000g, the pellet nuclei were collected. Fifty-microliter aliquots of the supernatants were frozen at -70°C, and the protein concentration was determined. DNA retardation (mobility shift) electrophoresis assays were performed as described by Fried and Crothers.²¹ Briefly, 5-µg nuclear extracts were incubated for 15 minutes in the presence of 5x binding buffer (125 mmol/L HEPES, pH 7.5, 50% glycerol, 250 mmol/L NaCl, 0.25% Nonidet P-40, 5 mmol/L DTT). An end-labeled, double-stranded consensus sequence of the murine-inducible NOS gene promoter nuclear factor–B (NF-B)–enhancing element and TNF-–responsive element (20 000 cpm per sample) was then added to the samples and incubated for 30 minutes. Samples were analyzed on a 5% nondenaturing polyacrylamide gel containing 0.01% Nonidet P-40. The specificity of the nuclear protein binding was assessed by incubating the nuclear proteins isolated from macrophages with the labeled DNA probe in the presence of a 1000-molar excess of unlabeled DNA probe.

DNA Probes
The cDNA probe for murine NOS was kindly provided by Dr D. Radzioch (McGill University, Montreal, Canada). The murine GAPDH probe was prepared in our laboratory by polymerase chain amplification. cDNA was obtained from total RNA by using a reverse-transcription reaction. Two synthetic primers were used to enzymatically amplify 456 bp of the GAPDH cDNA. The GAPDH probe was subsequently purified on a low-melting agarose gel. For Northern blot analysis, purified DNA inserts were labeled with [-³²P]dCTP by using a nick translation DNA labeling kit (Boehringer-Mannheim). A 20-mer double-stranded oligonucleotide (5'-GTGCTAGGGGGATTTTCCCT-3'; 5'-AGAGAGGGAAAATCCCCCTA-3') containing the consensus sequence for the NF-B enhancer of the murine NOS gene promoter and a 20-mer double-stranded oligonucleotide (5'-CGAGGCTGAGCTGACTTTGG-3'; 5'-GTCCCCAAAGTCAGCTCAGC-3') containing the TNF-–responsive element of the murine NOS gene promoter were synthesized with the aid of an automated DNA synthesizer. After annealing, the double-stranded oligonucleotides were labeled with [-³²P]ATP by using the Boehringer Mannheim 5'-end-labeling kit.

Determination of NO Production
The measure of NO produced by macrophages was performed according to the method of Green et al.²² Results were expressed as nitrite production per total protein content of macrophages.

Determination of Protein Concentrations
Total protein content was estimated according to the Bradford method²³ by using a colorimetric assay (BioRad).

	Results

Top Abstract Introduction Methods Results Discussion References

 
LPL Synergizes With IFN- to Induce Macrophage NO Production
We investigated the effect of LPL (1 µg/mL) alone or in combination with IFN- (100 U/mL) on macrophage NO production. ANA-1 macrophages were incubated with medium, IFN- (100 U/mL), or LPL (1 µg/mL) alone or in combination with IFN-, and NO production was determined in the medium after 24 hours. Macrophages did not produce NO either constitutively or after treatment with LPL alone. However, treatment of macrophages with LPL plus IFN- significantly increased IFN-–induced NO production (Fig 1, top). Treatment of LPL with 100 µg/mL polymyxin B did not modify the synergistic effect of LPL on IFN-–induced NO production (Fig 1, bottom). Heat-inactivation of LPL led to a slight decrease of IFN-–induced NO production but did not abolish the LPL effect, suggesting that LPL may, in part, exert its effect independently of its lipolytic activity. Furthermore, pancreatic lipase, which is structurally related to LPL but does not bind to heparin, was found to be as effective as LPL in inducing NO production (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 1. Bar graphs showing lipoprotein lipase (LPL) synergy with interferon gamma (IFN-) in the induction of macrophage nitric oxide (NO) production. Top, ANA-1 macrophages were incubated with medium (Med), LPL (1 µg/mL), or IFN- (100 U/mL) alone or in combination with LPL (1 µg/mL). NO accumulation was determined after 24 hours in the supernatants as described under "Methods." Data represent mean±SEM of six experiments; ***P<.01 vs control. Bottom, in some experiments, LPL was pretreated for 1 hour with (+) or without (-) polymyxin B (Poly; 100 µg/mL) and added to the cells in the presence of IFN- (100 U/mL). After 24 hours NO accumulation was determined in the supernatants as described under "Methods." Data represent mean±SEM of three experiments. IFN indicates IFN-.

To assess the specificity of the LPL effect on IFN-–induced NO production, LPL was preincubated for 1 hour at 37°C with an anti-LPL neutralizing antibody (10 µg/mL) and then added to the cells. Preincubation of LPL with anti-LPL antibody totally suppressed the synergistic effect of LPL on IFN-–induced NO formation (Fig 2).

View larger version (19K): [in this window] [in a new window]   Figure 2. Bar graph showing that anti–lipoprotein lipase (LPL) neutralizing antibody abolishes the LPL effect on interferon gamma (IFN-)–induced nitric oxide (NO) production. LPL was pretreated for 1 hour with or without an anti-LPL neutralizing antibody (10 µg/mL) and added to the cells in the presence of IFN- (100 U/mL). After 24 hours NO accumulation was determined in the supernatants as described under "Methods." Data represent mean±SEM of three experiments. Med indicates medium; IFN, IFN-. ***P<.01 vs control.

Synergistic Effect of LPL on IFN-–Induced NO Production Is LPL Dose Dependent
To determine the dose dependence of LPL effects, ANA-1 macrophages were incubated with increasing concentrations of LPL (0 to 5 µg/mL) in the presence or absence of IFN- (100 U/mL) for 24 hours. Treatment of the ANA-1 cells with LPL alone failed to induce NO production. In contrast, the combination of IFN- and increasing LPL concentrations enhanced the production of NO in an LPL dose–dependent manner comparatively to that observed with IFN- alone (Fig 3). The synergistic interaction between LPL and IFN- reached its maximum at a dose of 5 µg/mL LPL.

View larger version (49K): [in this window] [in a new window]   Figure 3. Bar graph showing dose-dependent effect of lipoprotein lipase (LPL) on interferon gamma (IFN-)–induced nitric oxide (NO) production. ANA-1 cells were incubated with LPL alone (0 to 5 µg/mL), IFN- alone (100 U/mL), or in combination with increasing LPL concentrations (0 to 5 µg/mL). After 24 hours NO accumulation was determined in the supernatants as described under "Methods." Data shown are from one representative experiment of three performed. IFN indicates IFN-.

To determine whether the sequence of the two signals could modify the extent of the synergistic interaction between LPL and IFN-, macrophages were preincubated for 1 hour with LPL alone and then treated with IFN- (100 U/mL) for an additional 24 hours. Pretreatment of ANA-1 cells with LPL did not induce higher levels of NO production than those observed when IFN- and LPL were added simultaneously (Fig 4). A slight but consistent augmenting production of NO was observed when the cells were pretreated with IFN- before being exposed to LPL (Fig 4).

View larger version (18K): [in this window] [in a new window]   Figure 4. Bar graph showing analysis of nitric oxide (NO) induction upon sequential treatment of ANA-1 cells with lipoprotein lipase (LPL) and interferon gamma (IFN-). Macrophages were incubated for 1 hour with medium (Med) or medium supplemented with 1 µg/mL LPL or 100 U/mL IFN-. After this incubation, LPL plus IFN-, IFN- alone, or LPL, respectively, were added to the cells for an additional 24 hours. NO accumulation was determined in the supernatants as described under "Methods." Data represent mean±SEM of three experiments. IFN indicates IFN-. *P<.05 vs control.

TNF- Is Involved in Stimulatory Effect of LPL on IFN-–Induced NO Secretion
Our previous observations showing a stimulatory effect of LPL on macrophage TNF- production next led us to verify the possibility that the synergistic effect of LPL on IFN-–induced NO production could be mediated by the release of TNF-. To test this hypothesis, supernatants from LPL-stimulated and unstimulated cells were collected after 24 hours, preincubated with an anti-murine TNF- neutralizing antibody, and then added for an additional 24-hour period of time to IFN-–stimulated macrophages. We found that macrophages exposed to LPL-treated supernatants produced higher amounts of NO than those exposed to control supernatants and that treatment of the LPL-treated supernatants with an anti–TNF- antibody totally abolished this effect (Fig 5). These data indicate that the LPL effects on IFN-–stimulated NO production are at least partially due to its ability to increase macrophage TNF- production.

View larger version (29K): [in this window] [in a new window]   Figure 5. Bar graph showing that the stimulatory effect of lipoprotein lipase (LPL) on interferon gamma (IFN-)–induced nitric oxide (NO) secretion requires tumor necrosis factor– (TNF-) secretion. Supernatants from unstimulated or LPL-stimulated ANA-1 cells were collected after 24 hours, preincubated in the presence of an anti-murine TNF- neutralizing antibody (anti-TNF) for 1 hour, and then added for an additional 24 hours to IFN-–stimulated macrophages. NO accumulation was determined in the supernatants as described under "Methods." Data represent mean±SEM of three experiments. Med indicates medium. *P<.05 vs control.

To further establish the role of TNF- as mediator of the LPL effects, we compared the levels of NO production by ANA-1 cells treated with TNF- or LPL in combination with IFN-. TNF- induced a similar increase of IFN-–induced macrophage NO production as that observed with LPL (Table). Although these results further strengthened the importance of TNF- in the observed LPL effects, they did not preclude the possibility that LPL could affect NO production by a TNF-–independent mechanism. This hypothesis was tested by incubating macrophages simultaneously with IFN-, LPL, and saturating concentrations of TNF-. The combination of all three agents led to a further increase of NO production compared with that observed in the presence of IFN- and LPL (Table). Overall, these data indicate that LPL may increase IFN-–induced NO production both by TNF-–dependent and –independent mechanisms.

View this table: [in this window] [in a new window]   Table 1. LPL Increases IFN-–Induced NO Production by Both TNF-–Dependent and –Independent Mechanisms

LPL Effect on NO Secretion Requires Activation of Macrophage-Inducible NOS
To evaluate the possibility that macrophages produce NO in an L-arginine–dependent manner upon stimulation with LPL, we tested the effect of N^G-methyl-L-arginine (L-NMMA), a specific inhibitor for NOS, on the LPL plus IFN-–induced NO formation. At a concentration of 1 mmol/L, this arginine analogue completely abolished the LPL effect on IFN-–induced NO secretion (Fig 6). This effect was still observable with a concentration of L-NMMA as low as 0.1 mmol/L (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 6. Bar graph showing that L-arginine–dependent nitric oxide (NO) synthetase activity is required for lipoprotein lipase (LPL) effects on NO production. ANA-1 macrophages were incubated in the presence or absence of aminoguanidine (1 mmol/L), with medium (Med), LPL (1 µg/mL), or interferon gamma (IFN) (100 U/mL) alone or in combination with LPL (1 µg/mL). After 24 hours NO accumulation was determined in the supernatants as described under "Methods." Data represent mean±SEM of three experiments.

LPL Synergizes With IFN- to Induce Macrophage NOS Gene Expression
To investigate the mechanisms by which LPL increased IFN-–dependent NO production, we evaluated the effects of LPL on macrophage NOS mRNA expression. ANA-1 cells were exposed for 6, 12, 18, or 24 hours to 100 U/mL IFN- in the presence or absence of 1 µg/mL LPL. A synergistic effect of LPL and IFN- on NOS mRNA expression was observed, the peak in NOS mRNA expression occurring at 18 hours (Fig 7).

View larger version (59K): [in this window] [in a new window]   Figure 7. Autoradiographs showing that lipoprotein (LPL) synergizes with interferon gamma (IFN-) to induce macrophage nitric oxide synthetase (NOS) gene expression. ANA-1 macrophages were incubated with medium (M), LPL (1 µg/mL), or IFN- (100 U/mL) alone or in combination with LPL (1 µg/mL) for the indicated times. Total cellular RNA was analyzed for NOS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression as described under "Methods." IFN indicates IFN-.

Protein Synthesis Is Required for NOS mRNA Induction by IFN- Plus LPL
To establish whether LPL-induced NOS mRNA expression was dependent on de novo protein synthesis, ANA-1 macrophages were incubated with medium, IFN-, or LPL alone or in combination with IFN- in the presence or absence of CHX (7.5 µg/mL) for 6 hours. Addition of CHX totally abolished the induction of NOS mRNA by LPL in combination with IFN- (Fig 8A). These results show that de novo protein synthesis is required for NOS mRNA induction by LPL plus IFN-.

View larger version (29K): [in this window] [in a new window]   Figure 8. Autoradiographs. A, De novo synthesis of protein is required for nitric oxide synthetase (NOS) mRNA induction by interferon gamma (IFN-) plus lipoprotein lipase (LPL). ANA-1 macrophages were incubated with medium (M), IFN- (IFN), or LPL alone or in combination with IFN- in the presence or absence of cycloheximide (CHX; 7.5 µg/mL) for 6 hours. Total cellular RNA was analyzed for NOS mRNA expression as described under "Methods." B, Tumor necrosis factor– (TNF-) is involved in the synergistic effect of LPL on IFN-–induced NOS mRNA expression. Supernatants from unstimulated or LPL-stimulated ANA-1 cells were collected after 24 hours, preincubated in the presence of an anti-murine TNF- neutralizing antibody for 1 hour, and added for an additional 18 hours to IFN-–stimulated macrophages. Total cellular RNA was analyzed for NOS mRNA expression as described under "Methods."

TNF- Mediates LPL Effect on NOS mRNA Expression
To establish the role of TNF- in the LPL-induced NOS mRNA expression, supernatants from LPL-stimulated and unstimulated cells were collected after 24 hours of treatment, preincubated with an anti-murine TNF- neutralizing antibody, and then added for an additional 24 hours to IFN-–stimulated macrophages. Macrophages exposed to LPL-treated supernatants expressed higher NOS mRNA levels than those exposed to control supernatants (Fig 8B). Treatment of the LPL-treated supernatants with an anti–TNF- antibody totally abolished the stimulatory effect of LPL on NOS mRNA expression (Fig 8B). These results indicate that TNF- is at least partly responsible for the stimulatory effect of LPL on macrophage NOS mRNA expression.

IFN- Plus LPL Induces Transcriptional Activation of NOS
To investigate the mechanism(s) by which LPL increased NOS mRNA expression, we performed a nuclear run-on assay. ANA-1 cells were incubated with medium, IFN-, LPL, or IFN- plus LPL, and the nuclei were isolated after 18 hours of treatment. IFN- alone induced very low levels of transcription, whereas the combination of IFN- with LPL caused a significant increase in the rate of transcription of the NOS gene (Fig 9). These data indicate that LPL in combination with IFN- augments the transcriptional activity of the NOS gene.

View larger version (27K): [in this window] [in a new window]   Figure 9. Blot showing effect of lipoprotein lipase (LPL) plus interferon gamma (IFN-) on nitric oxide synthetase (NOS) gene transcription. ANA-1 macrophages were incubated with medium (M), LPL (1 µg/mL), or IFN- (100 U/mL) alone or in combination with LPL (1 µg/mL). Nuclei were isolated after 18 hours, and the rate of transcription of the NOS gene was determined by nuclear run-on analysis as described under "Methods." A pBluescript (pBS) vector lacking a cDNA insert was used as a control for nonspecific hybridization. GAPDH indicates glyceraldehyde-3-phosphate dehydrogenase; IFN, IFN-.

LPL Induces Changes at the Level of Macrophage NOS Gene-Promotor Binding Proteins
To test whether treatment of macrophages with LPL could induce changes at the level of NOS gene-promoter binding, nuclear proteins isolated from untreated cells and LPL-, IFN-–, and LPL plus IFN-–treated macrophages were tested for binding to the NF-B– and TNF-–responsive consensus sequences described in the IFN-–responsive region of the murine macrophage NOS promotor. Decreased nuclear protein binding to these regulatory sequences was observed in IFN-– and LPL plus IFN-–treated macrophages compared with that observed in untreated or LPL-treated cells (Fig 10). The specificity of the protein binding was assessed by incubating the nuclear proteins isolated from LPL plus IFN-–treated macrophages with the labeled DNA probes in the presence of a 1000-molar excess of unlabeled DNA probes.

View larger version (48K): [in this window] [in a new window]   Figure 10. Autoradiograph showing that lipoprotein lipase (LPL) plus interferon gamma (IFN-) decreases the nuclear protein binding to the regulatory nuclear factor–B (NF-B) and tumor necrosis factor regulatory element (TNF-RE) sequences of the nitric oxide synthetase (NOS) gene promoter. ANA-1 cells were treated for 18 hours with medium (M), LPL (1 µg/mL), or IFN- (100 U/mL) alone or in combination with LPL (1 µg/mL). The nuclear proteins isolated from the cells were incubated with double-stranded NF-B and TNF regulatory elements of the NOS gene promoter. The specificity of the protein binding to both consensus sequences was demonstrated by competition (COMP.) experiments. Retardation was assessed by gel electrophoresis in 5% polyacrylamide gel. IFN indicates IFN-.

Effect of LPL on NOS mRNA Stability
To establish whether LPL may also act at the posttranscriptional level, ANA-1 macrophages were treated with IFN- alone or in combination with LPL for 18 hours. Actinomycin D (5 µg/mL) was then added to the culture, total RNA was harvested at different times, and the half-life of the NOS mRNA was monitored by Northern blot analysis for 6 hours. We observed no significant differences in the half-life of NOS mRNA (approximately 3.5 hours) regardless of the presence or absence of LPL (data not shown). These results indicate that LPL exerts its stimulatory effect on IFN-–induced NOS mRNA expression at the transcriptional level.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
LPL is a glycoprotein constitutively secreted by macrophages.⁷ This enzyme plays a crucial role in plasma lipoprotein processing and energy metabolism.⁸ LPL increases the binding of VLDL and LDL to macrophages, thus enhancing their uptake of triglyceride-rich lipoproteins.^{24 25} In addition to its ability to affect macrophage lipid metabolism, LPL induces macrophage TNF- production,¹³ which involves the activation of protein kinase C.²⁶ Murine macrophages respond to IFN- with low levels of NO production.² A combination of IFN- and TNF- induces macrophage NO-dependent microbicidal activity.²⁷

The ability of LPL to induce TNF- production led us to evaluate the effects of LPL on macrophage NO production. LPL, although ineffective by itself, acted synergistically with IFN- to induce macrophage NO production. These results, together with our previous observations of LPL-induced macrophage TNF- production, further establish the role of LPL in the control of macrophage activation.

To rule out the possibility that the LPL effect could be due to the presence of endotoxin in our LPL preparations, we measured the levels of endotoxin in the LPL samples. We consistently found that the content of endotoxin was below the detection limit of 6 pg/mL in all the LPL preparations tested. The specificity of the LPL effect was also tested by incubating LPL in the presence of polymyxin B or in the presence of a neutralizing LPL antibody before its addition to the cells. Incubation of LPL with polymyxin B did not abolish its effect, whereas the anti-LPL antibody totally abrogated the synergistic effect of LPL on IFN-–induced NO production.

The ability of heat-inactivated LPL to increase IFN-–induced NO production suggests that LPL may exert its effect at least partly independently of its lipolytic activity. These results and our observation that pancreatic lipase, which does not bind to heparin, was as effective as LPL in inducing NO production support the possibility that lipolytic and heparin-binding properties are not essential for the effect of LPL on NO production.

IFN- represents a warning signal that evokes a transient level of readiness in the macrophage, so-called "priming," but which requires further potentiation to induce full activation of the cell. In the case of NO production, cooperation between IFN- and lipopolysaccaride (LPS) does not require a strict hierarchical order.²⁸ Similarly, we report that the temporal sequence of exposure of the cells to each single stimulus is not essential to obtain an optimal synergistic interaction between LPL and IFN-, although the increase in NO production was slightly more pronounced when IFN- was the first stimulus.

The presence of an anti–TNF- antibody in cultures of LPS-stimulated macrophages partially inhibits the macrophage-dependent NO production induced by IFN- and LPS.²⁹ Our previous observation that LPL induces macrophage TNF- production suggested that LPL might trigger macrophage NO formation via the induction of TNF-. Our results support this hypothesis by demonstrating that the addition of an anti–TNF- antibody to supernatants of LPL-treated macrophages abrogates the ability of LPL to synergize with IFN- for the induction of NO production. This finding at least partially explains how LPL can cooperate with IFN- to achieve high macrophage NO secretion. However, these results do not exclude the possibility that TNF-–independent mechanism(s) might also be involved in the induction of NO production by IFN- plus LPL. To test this possibility, we evaluated the ability of LPL to increase NO production by macrophages treated with IFN- and high TNF- concentrations. The further increase of NO secretion we observed under these experimental conditions indicates that TNF-–independent mechanism(s) could also be responsible for the LPL effects. Whether LPL may act directly to exert its stimulatory effect on NO production remains to be established.

The main rate-limiting step for NO production by macrophages is the intracellular levels of inducible NOS. NOS enzymatic activity is efficiently induced within a few hours after stimulation of the cells with LPS or IFN-.^{30 31} Evidence for a role of NOS in the observed LPL effects is provided by the significant inhibition of NO production observed in LPL-treated macrophages cultured in presence of the specific NOS inhibitor L-NMMA. These data indicate that inducible L-arginine–dependent NOS activity is required for the LPL effects on NO production.

Activation of NOS gene expression occurs after macrophage stimulation with IFN- and LPS.²⁸ We report in this study that LPL synergizes with IFN- to induce NOS mRNA expression, thus providing evidence for the molecular basis of the synergistic interaction between IFN- and LPL in the generation of NO production.

To elucidate the mechanisms involved in the induction of NOS mRNA by LPL, ANA-1 cells were stimulated with IFN- plus LPL in presence of CHX, an inhibitor of protein synthesis. Pretreatment of the cells with CHX completely abolished the accumulation of NOS mRNA in response to LPL plus IFN-. These results demonstrate that de novo protein synthesis is required for NOS mRNA induction by LPL plus IFN- and suggest that LPL may enhance the expression of the NOS gene via the induction of an intermediary protein or proteins.

Since TNF- acts as a mediator of the LPL effects on macrophage NO production, we tested whether TNF- may represent a regulatory factor involved in LPL-dependent induction of the macrophage NOS gene. The abrogation of the synergistic effect of LPL on IFN-–induced NOS mRNA expression observed in the presence of an anti–TNF- antibody indicates that TNF- represents one intermediary protein involved in the activation of NOS gene expression by LPL.

Activation of NOS gene expression by IFN- plus LPS occurs at the transcriptional level.^{32 33} Nuclear run-on experiments demonstrated that LPL plus IFN- increased the rate of transcription of the NOS gene, providing evidence that LPL can exert transcriptional control of NOS gene expression.

The recent identification in the promoter region of the mouse NOS gene of potential response elements for regulatory transcription factors led us to further evaluate the role of DNA-binding transcription factors in the LPL-stimulated activation of the NOS gene. Two potential binding sites for NF-B and for a transcription factor motif that binds factors associated with stimulation by TNF- (TNF-responsive elements) have been localized to two discrete regions involved in regulating the expression of the mouse NOS gene in response to IFN- and LPS.³⁴ Our previous observations that the NF-B–type enhancer element is involved in LPL-mediated transcriptional activation of the TNF- gene¹³ and the demonstration of a role of TNF- in the induction of NOS gene expression by LPL led us to verify the possibility that LPL may affect the binding of nuclear proteins to the NF-B– and TNF-responsive elements. The consistent decrease of protein binding to both sequences upon IFN- and LPL plus IFN- stimulation reported here may suggest that in resting macrophages repressor proteins could prevent the transcription of the NOS gene and that treatment with IFN- alone or in combination with LPL may stop these repressors from binding, leading to an enhanced transcription of the NOS gene.

Studies on the posttranscriptional effect of LPS on NOS mRNA stability conflict; one study shows that LPS can prevent IFN-–induced destabilization of this RNA,²⁸ but another fails to observe any posttranscriptional effect of this agent on NOS mRNA stability. Our inability to demonstrate any increase of NOS mRNA stability after LPL plus IFN- treatment suggests that the posttranscriptional effect of LPL on NOS mRNA does not account for the accumulation of NOS mRNA upon LPL stimulation.

The in vivo relevance of our observations remains uncertain. While it is not possible to estimate the concentration of LPL in the macrophage interstitial milieu, cultured human monocyte–derived macrophages can produce 40 ng/mL LPL daily.³⁵ In the present study the levels needed to achieve significant stimulation of NO production levels were much higher than the levels cultured macrophages may produce endogenously. The constitutive macrophage LPL production can, however, be significantly enhanced under different conditions. Agents that affect cell proliferation and differentiation³⁶ or receptor-mediated endocytosis of ligands via the scavenger receptor³⁷ markedly increase macrophage LPL secretion. LPL production is also increased in inflammatory and primed macrophages.³⁸ Since other cell types besides the monocyte/macrophage, eg, smooth muscle cells, produce LPL, one may postulate that under some conditions, such as the atherosclerotic process, the massive accumulation of macrophages and smooth muscle cells and the local expression in the arterial wall of LPL stimulatory factors such as macrophage-colony stimulating factor could generate sufficiently high local LPL concentrations to influence macrophage biology.

In conclusion, our study demonstrates a synergistic effect of LPL on IFN-–induced macrophage NOS expression and NO secretion and provides evidence for a role of TNF- as mediator of the observed LPL effects. These results further emphasize the critical role of LPL as an autocrine activator of macrophage function.

	Acknowledgments

 
We thank Dr Juan DeSanctis (Institute of Immunology, Caracas, Venezuela) for providing the purified human LPL and the anti-LPL antibody.

Received September 9, 1994; accepted December 12, 1994.

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