Bioactive products generated by group V sPLA(2) hydrolysis of LDL activate macrophages to secrete pro-inflammatory cytokines.
Journal: 2010/May - Cytokine
ISSN: 1096-0023
Abstract:
OBJECTIVE
Previous studies have established that hydrolysis of LDL by Group V secretory phospholipase A(2) (GV sPLA(2)) generates a modified particle capable of inducing macrophage foam cell formation. The aim of the present study was to determine whether GV sPLA(2)-hydrolyzed LDL (GV-LDL) produces pro-atherogenic effects in macrophages independent of cholesterol accumulation.
RESULTS
J-774 cells incubated with GV-LDL produced more TNF-alpha and IL-6 compared to cells incubated with control-LDL. Indirect immunofluorescence showed that GV-LDL but not control-LDL induced nuclear translocation of NFkappaB. Inhibitors of NFkappaB activation, effectively blocked cytokine production induced by GV-LDL. Control-LDL and GV-LDL were separated from albumin present in reaction mixtures by ultracentrifugation. The albumin fraction derived from GV-LDL contained 80% of the FFA generated and was more potent than the re-isolated GV-LDL in inducing pro-inflammatory cytokine secretion. Linoleic acid (18:2) and oleic acid (18:1) were the most abundant FFAs generated, whereas newly formed lyso-PCs contained 14:0 (myristic), 16:1 (palmitic), and 18:2 fatty acyl groups. Experiments with synthetic FFA showed that 18:1 induced J-774 cells to secrete TNF-alpha and IL-6.
CONCLUSIONS
These results indicate that in addition to promoting atherosclerotic lipid accumulation in macrophages, GV sPLA(2) hydrolysis of LDL leads to activation of NFkappaB, a key regulator of inflammation.
Relations:
Content
Citations
(8)
References
(44)
Chemicals
(8)
Genes
(3)
Organisms
(4)
Processes
(5)
Anatomy
(3)
Affiliates
(1)
Similar articles
Articles by the same authors
Discussion board
Cytokine 50(1): 50-57

Bioactive products generated by group V sPLA<sub>2</sub> hydrolysis of LDL activate macrophages to secrete pro-inflammatory cytokines

Objective

Previous studies have established that hydrolysis of LDL by Group V secretory phospholipase A2 (GV sPLA2) generates a modified particle capable of inducing macrophage foam cell formation. The aim of the present study was to determine whether GV sPLA2-hydrolyzed LDL (GV-LDL) produces pro-atherogenic effects in macrophages independent of cholesterol accumulation.

Methods and Results

J-774 cells incubated with GV-LDL produced more TNF-α and IL-6 compared to cells incubated with control-LDL. Indirect immunofluorescence showed that GV-LDL but not control-LDL induced nuclear translocation of NFκB. Inhibitors of NFκB activation, effectively blocked cytokine production induced by GV-LDL. Control-LDL and GV-LDL were separated from albumin present in reaction mixtures by ultracentrifugation. The albumin fraction derived from GV-LDL contained 80% of the FFA generated and was more potent than the re-isolated GV-LDL in inducing pro-inflammatory cytokine secretion. Linoleic acid (18:2) and oleic acid (18:1) were the most abundant FFAs generated, whereas newly formed lyso-PCs contained 14:0 (myristic), 16:1 (palmitic), and 18:2 fatty acyl groups. Experiments with synthetic FFA showed that 18:1 induced J-774 cells to secrete TNF-α and IL-6.

Conclusions

These results indicate that in addition to promoting atherosclerotic lipid accumulation in macrophages, GV sPLA2 hydrolysis of LDL leads to activation of NFκB, a key regulator of inflammation.

1. Introduction

According to current paradigms, the development of atherosclerosis culminates from a gradual increase in lipid deposition and chronic inflammation in the subendothelium [1-2]. The subendothelial retention of atherogenic lipoproteins provides substrate for multiple modifying agents. These modified lipoproteins are subsequently taken up by locally recruited macrophages to form foam cells, which in turn are activated to produce various cytokines that promote and maintain local inflammation.

The secretory phospholipase A2 (sPLA2) family of enzymes has been implicated in pro-atherosclerotic processes. The sPLA2's hydrolyze the ester bond at the sn-2 position of glycerophospholipids to release free fatty acids (FFA) and lyso-phospholipids (lyso-PL) [3-4]. Several lines of evidence suggest that sPLA2's play a role in atherosclerosis through their hydrolyzing activities in the arterial intima [5-7]. To date, seven members of the sPLA2 family have been detected in atherosclerotic lesions [8]. Of these, group V (GV), group X (GX) and recently Group III (GIII) have been shown to effectively hydrolyze LDL phospholipids (PL) in vitro [9-11]. Hydrolysis of LDL by sPLA2 leads to alterations in the conformation of apoB100 on the PL-depleted particle, which destabilizes the particle and promotes aggregation. The structurally altered LDL particle also exhibits enhanced binding to extracellular matrix and cell-surface proteoglycans [12], which is likely due to the exposure of a proteoglycan binding site present on apoB-100 that is normally buried within the LDL particle [13]. In the case of GV sPLA2-modified LDL (GV-LDL), recent data indicate that syndecan 4, a proteoglycan expressed on the surface of macrophages, mediates uptake of GV-LDL to form foam cells [14]. Thus, based on numerous in vitro studies, LDL hydrolysis by sPLA2 may promote atherosclerosis by enhancing the retention of LDL particles in the subendothelium and by promoting macrophage LDL uptake.

The possibility that GV sPLA2 promotes atherosclerotic lipid deposition in vivo is supported by gain-of-function and loss-of-function studies carried out in LDL receptor mice [15]. However, in studies in apoE mice, deficiency of GV sPLA2 had no effect on atherosclerotic lesion area in either male or female mice [16]. The discrepancy in results from the two mouse models may be partially explained by in vitro studies showing that GV sPLA2 modification of LDL from LDL receptor mice enhances the capacity of the particle to promote macrophage foam cell formation, whereas GV sPLA2 modification of LDL from apoE mice lacked this pro-atherogenic effect. Interestingly, apoE × GV sPLA2 double knock-out mice had significantly less collagen deposition in lesions compared to apoE mice despite similar atherosclerotic lipid area. Thus, GV sPLA2 activity in the arterial intima may lead to two independent processes: 1) macrophage foam cell formation through the generation of a structurally altered particle; and 2) altered gene expression through the generation of bioactive lipid mediators.

The objective of this study was to investigate whether GV-LDL produces inflammatory effects in macrophages independent of cholesterol accumulation. Although there is data suggesting that PLA2s and their lipolytic products, namely lyso-PL and FFA, modulate inflammation and thus have an effect on atherosclerosis development, the current literature is controversial. For example, Curfs et al. have reported that GX sPLA2 has anti-inflammatory effects in vitro, however its overexpression in vivo triggers lung pathology consistent with massive inflammatory cell deposition [17]. Studies showing that saturated FFA but not unsaturated fatty acids activate NFκB in macrophages by stimulating TLR4 [18-19] have been recently questioned [20]. Lyso-PCs have been implicated in pro-inflammatory responses in neural tissue [21] but on the other hand have been shown to abrogate effects of lipopolysaccharide (LPS) in neutrophils [22].

In the current study we show that lipolytic products released from GV-LDL induce NFκB activation in macrophages, and consequently, the secretion of pro-inflammatory cytokines. Thus, this study provides an additional mechanism by which sPLA2 may promote atherogenic processes.

Experimental procedures

2.1 Isolation and modification of LDL

LDL (density 1.019-1.063) was isolated from the plasma of healthy volunteers by sequential ultracentrifugation and stored at 4 °C under argon gas. For LDL hydrolysis, mouse GV sPLA2 was partially purified from conditioned media of COS-7 cells infected with a replication-defective adenoviral vector encoding this enzyme as described previously [10]. Phospholipase activity of partially purified enzyme was determined using a colorimetric assay with 1-palmitoyl-2-oleoyl-phosphatidyglycerol (POPG) as substrate (1 unit generates 1 nmol of FFA in 20 minutes) [10]. LDL particles (1 mg/ml) were incubated overnight in hydrolysis buffer (0.1 M HEPES, 0.1 M NaCl, 1 mM CaCl2, 20 mg/ml fatty acid-free BSA and 0.01% butylated hydroxytoluene) under argon gas at 37 °C in the presence of 500 U/ml GV sPLA2 (GV-LDL). LDL hydrolysis was quantified by measuring the amount of FFA released in the solution [10]. As control, LDL was incubated overnight in hydrolysis buffer in the absence of the enzyme (control-LDL). For the experiments described here, incubation with GV sPLA2 resulted in the hydrolysis of ∼ 60% of LDL-PL. As reported previously [23], there was no evidence that GV sPLA2 hydrolysis led to apoB degradation or alterations in the charge of LDL particles. To remove large aggregates, GV-LDL hydrolysis mixtures were passed through a 0.1-μm filter. Details regarding density gradient ultracentrifugation of control-LDL and GV-LDL and the characterization of fractions derived from control-LDL and GV-LDL are provided in Supplemental Materials.

2.2 Cell culture

J-774 macrophage-like cells were obtained from the American Type Culture Collection. The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin, 2 mM L-glutamine. Cells were incubated for the indicated times with 100 μg/ml control-LDL or GV-LDL or 2 mg/ml albumin-enriched fraction derived from control-LDL and GV-LDL. Throughout the experiments 100 ng/ml lipopolysaccharide (LPS) was used as positive control. When indicated, cells were pre-incubated for 1 h with 20 μM of the specific NFκB inhibitor SN50 or a control peptide (Calbiochem) or 10 μM 6-amino-4-phenethylaminoquinazoline, an inhibitor of NFκB transcriptional activation(Calbiochem) [24]. All reagents besides LPS were tested for endotoxin contamination using the limulus amebocyte lysate assay (Cambrex) and were below the limits of detection (< 0.1 ng/ml). For treatments with oleic and linoleic acid, preparation of experimental media was carried out as described by Toborek et al. [25]. Stock solutions of high purity (>99%) fatty acids (Nu-Chek-Prep, Elysian, MN) were prepared in hexane. NaOH (6 N, or 30× molarity of FFA) was used for saponification to convert the FFA into a water-soluble form. The desired amount of FFA was aliquoted, mixed with 6 M NaOH, and dried under argon gas. The residue was dissolved in experimental medium containing 5% FBS. The pH was then adjusted to 7.4 with 1.2 M HCl and the medium was sterilized through a syringe-driven filter unit.

2.3 Real-Time RT- PCR

Total RNA was isolated from J-774 cells using the TriReagent (Molecular Research Center, Inc.). RNA (1-2 μg) was reverse transcribed into cDNA using the Reverse-transcription System (Promega). After 4-fold dilution, 5 μl was used as a template for real-time PCR. Amplification was done for 40 cycles using Power SYBR Green PCR master Mix Kit (Applied Biosystems) and DNA Engine Optical 2 System (MJ Research Inc.). Quantification was performed in duplicate using the standard curve method and normalizing to 18S. The primers used for various genes are as follows (gene: primers): mTNF-α forward: 5′-GGCAGGTCTACTTTGGAGTCATTG-3′, reverse: 5′-GGTAGAAGGACACAGACTGG-3′; mIL-6 forward:5′-CAACGATGATGCACTTGCAG-3′, reverse: 5′-GTAGCTATGGTACTCCAGAAG-3′; m18S RNA, forward: 5′-GCTGGAATTACCGCGGCT-3′; reverse: 5′-CGGCTACCACATCCAAGGAA-3′.

2.4 TNF-α and IL-6 measurements

TNF-α and IL-6 were quantified using enzyme linked immunosorbent assay (ELISA) kits (R&amp;D Systems).

2.5 Indirect immunofluorescence

Cells were grown on glass cover-slips for confocal microscopy. After the treatments, J-774 cells were fixed in 10% formaldehyde for 10 min, permeabilized with 0.1 M Na-citrate for 15 min and then subjected to indirect immunofluorescent staining using mouse anti-p65 IgG (Santa Cruz). Alexa Fluor-488 labeled goat anti-mouse IgG (Molecular Probes) was used as secondary antibody at a dilution of 1:200. As control, cells were incubated in the presence of non-specific primary antibody. Cellular cytoskeleton was visualized with rhodamine phalloidin staining (R415, Molecular Probes) at room temperature for 1 h. After incubations, cells were washed extensively and mounted on slides using Fluorescence Protecting Medium containing DAPI (Vectashield, Vector Llaboratories, Burlingam, CA). Confocal microscopy was performed at the University of Kentucky Imaging Facility using a Leica laser scanning confocal microscope with argon (488 nm) and krypton (568 nm) lasers. Nuclear localization of NFκB was quantified in at least 50 randomly chosen cells using ImagePro™ software. All areas with positive staining for p65 were selected and fluorescent area associated with the nucleus (defined by DAPI staining) was expressed as % of the total fluorescent area in cells (defined by phalloidin staining).

2.6 Quantitation of phospholipids and fatty acids by HPLC ESI tandem Mass spectrometry

Lipids and fatty acids were extracted from LDL and BSA fractions using acidified organic solvents [26] with addition of 50 pmol C17 lysoPC as an internal recovery standard. After evaporation to dryness material was dissolved in 1 ml 4:1 MeOH:CHCl3 and an aliquot removed for phosphorous determination after wet digestion in perchloric acid. Half of the remaining material was used for phospholipid analysis. Molecular species of PC and lyso-PC were quantified by reverse phase HPLC electrospray ionization tandem mass spectrometry using an ABI 4000 Q-Trap hybrid linear ion trap triple quadrupole mass spectrometer operated in positive mode with ion source and instrument settings optimized using a set of internal standards [27-28]. Molecular species specific precursor product transitions were monitored and calibration accomplished by reference to a set of synthetic standards obtained from Avanti Polar Lipids that were independently quantified by phosphorous analysis.

Fatty acids were quantified as 3-acyloxymethyl-1-methylpyridinium iodide (AMMP) derivatives using a modification of the method described by W-C Yang et al. [28]. In brief, the remaining half of the extracted material was evaporated to dryness, derivatized by reaction with 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide methyl pyridinium and the AMPP derivatives analysed by HPLC ESI tandem mass spectrometry using an ABI 4000 Q-Trap hybrid linear ion trap triple quadrupole mass spectrometer operated in positive mode with ion source and instrument settings optimized using a set of AMPP derivatized fatty acid standards. Fatty acid molecular species were quantified using specific precursor product ion transitions. Calibration was accomplished by reference to a set of synthetic fatty acid AMPP derivatives prepared from accurate mass standards obtained from NuCheck Prep. Product ion spectra were generated for the most abundant species to determine the position of double bonds.

2.7 Statistical analysis

Data are expressed as mean ± SE. Results were analyzed by one or two-way ANOVA followed by Tukey's or Bonferroni's post-test, respectively. Values of P<0.05 were considered statistically significant.

2.1 Isolation and modification of LDL

LDL (density 1.019-1.063) was isolated from the plasma of healthy volunteers by sequential ultracentrifugation and stored at 4 °C under argon gas. For LDL hydrolysis, mouse GV sPLA2 was partially purified from conditioned media of COS-7 cells infected with a replication-defective adenoviral vector encoding this enzyme as described previously [10]. Phospholipase activity of partially purified enzyme was determined using a colorimetric assay with 1-palmitoyl-2-oleoyl-phosphatidyglycerol (POPG) as substrate (1 unit generates 1 nmol of FFA in 20 minutes) [10]. LDL particles (1 mg/ml) were incubated overnight in hydrolysis buffer (0.1 M HEPES, 0.1 M NaCl, 1 mM CaCl2, 20 mg/ml fatty acid-free BSA and 0.01% butylated hydroxytoluene) under argon gas at 37 °C in the presence of 500 U/ml GV sPLA2 (GV-LDL). LDL hydrolysis was quantified by measuring the amount of FFA released in the solution [10]. As control, LDL was incubated overnight in hydrolysis buffer in the absence of the enzyme (control-LDL). For the experiments described here, incubation with GV sPLA2 resulted in the hydrolysis of ∼ 60% of LDL-PL. As reported previously [23], there was no evidence that GV sPLA2 hydrolysis led to apoB degradation or alterations in the charge of LDL particles. To remove large aggregates, GV-LDL hydrolysis mixtures were passed through a 0.1-μm filter. Details regarding density gradient ultracentrifugation of control-LDL and GV-LDL and the characterization of fractions derived from control-LDL and GV-LDL are provided in Supplemental Materials.

2.2 Cell culture

J-774 macrophage-like cells were obtained from the American Type Culture Collection. The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin/streptomycin, 2 mM L-glutamine. Cells were incubated for the indicated times with 100 μg/ml control-LDL or GV-LDL or 2 mg/ml albumin-enriched fraction derived from control-LDL and GV-LDL. Throughout the experiments 100 ng/ml lipopolysaccharide (LPS) was used as positive control. When indicated, cells were pre-incubated for 1 h with 20 μM of the specific NFκB inhibitor SN50 or a control peptide (Calbiochem) or 10 μM 6-amino-4-phenethylaminoquinazoline, an inhibitor of NFκB transcriptional activation(Calbiochem) [24]. All reagents besides LPS were tested for endotoxin contamination using the limulus amebocyte lysate assay (Cambrex) and were below the limits of detection (< 0.1 ng/ml). For treatments with oleic and linoleic acid, preparation of experimental media was carried out as described by Toborek et al. [25]. Stock solutions of high purity (>99%) fatty acids (Nu-Chek-Prep, Elysian, MN) were prepared in hexane. NaOH (6 N, or 30× molarity of FFA) was used for saponification to convert the FFA into a water-soluble form. The desired amount of FFA was aliquoted, mixed with 6 M NaOH, and dried under argon gas. The residue was dissolved in experimental medium containing 5% FBS. The pH was then adjusted to 7.4 with 1.2 M HCl and the medium was sterilized through a syringe-driven filter unit.

2.3 Real-Time RT- PCR

Total RNA was isolated from J-774 cells using the TriReagent (Molecular Research Center, Inc.). RNA (1-2 μg) was reverse transcribed into cDNA using the Reverse-transcription System (Promega). After 4-fold dilution, 5 μl was used as a template for real-time PCR. Amplification was done for 40 cycles using Power SYBR Green PCR master Mix Kit (Applied Biosystems) and DNA Engine Optical 2 System (MJ Research Inc.). Quantification was performed in duplicate using the standard curve method and normalizing to 18S. The primers used for various genes are as follows (gene: primers): mTNF-α forward: 5′-GGCAGGTCTACTTTGGAGTCATTG-3′, reverse: 5′-GGTAGAAGGACACAGACTGG-3′; mIL-6 forward:5′-CAACGATGATGCACTTGCAG-3′, reverse: 5′-GTAGCTATGGTACTCCAGAAG-3′; m18S RNA, forward: 5′-GCTGGAATTACCGCGGCT-3′; reverse: 5′-CGGCTACCACATCCAAGGAA-3′.

2.4 TNF-α and IL-6 measurements

TNF-α and IL-6 were quantified using enzyme linked immunosorbent assay (ELISA) kits (R&amp;D Systems).

2.5 Indirect immunofluorescence

Cells were grown on glass cover-slips for confocal microscopy. After the treatments, J-774 cells were fixed in 10% formaldehyde for 10 min, permeabilized with 0.1 M Na-citrate for 15 min and then subjected to indirect immunofluorescent staining using mouse anti-p65 IgG (Santa Cruz). Alexa Fluor-488 labeled goat anti-mouse IgG (Molecular Probes) was used as secondary antibody at a dilution of 1:200. As control, cells were incubated in the presence of non-specific primary antibody. Cellular cytoskeleton was visualized with rhodamine phalloidin staining (R415, Molecular Probes) at room temperature for 1 h. After incubations, cells were washed extensively and mounted on slides using Fluorescence Protecting Medium containing DAPI (Vectashield, Vector Llaboratories, Burlingam, CA). Confocal microscopy was performed at the University of Kentucky Imaging Facility using a Leica laser scanning confocal microscope with argon (488 nm) and krypton (568 nm) lasers. Nuclear localization of NFκB was quantified in at least 50 randomly chosen cells using ImagePro™ software. All areas with positive staining for p65 were selected and fluorescent area associated with the nucleus (defined by DAPI staining) was expressed as % of the total fluorescent area in cells (defined by phalloidin staining).

2.6 Quantitation of phospholipids and fatty acids by HPLC ESI tandem Mass spectrometry

Lipids and fatty acids were extracted from LDL and BSA fractions using acidified organic solvents [26] with addition of 50 pmol C17 lysoPC as an internal recovery standard. After evaporation to dryness material was dissolved in 1 ml 4:1 MeOH:CHCl3 and an aliquot removed for phosphorous determination after wet digestion in perchloric acid. Half of the remaining material was used for phospholipid analysis. Molecular species of PC and lyso-PC were quantified by reverse phase HPLC electrospray ionization tandem mass spectrometry using an ABI 4000 Q-Trap hybrid linear ion trap triple quadrupole mass spectrometer operated in positive mode with ion source and instrument settings optimized using a set of internal standards [27-28]. Molecular species specific precursor product transitions were monitored and calibration accomplished by reference to a set of synthetic standards obtained from Avanti Polar Lipids that were independently quantified by phosphorous analysis.

Fatty acids were quantified as 3-acyloxymethyl-1-methylpyridinium iodide (AMMP) derivatives using a modification of the method described by W-C Yang et al. [28]. In brief, the remaining half of the extracted material was evaporated to dryness, derivatized by reaction with 2-bromo-1-methylpyridinium iodide and 3-carbinol-1-methylpyridinium iodide methyl pyridinium and the AMPP derivatives analysed by HPLC ESI tandem mass spectrometry using an ABI 4000 Q-Trap hybrid linear ion trap triple quadrupole mass spectrometer operated in positive mode with ion source and instrument settings optimized using a set of AMPP derivatized fatty acid standards. Fatty acid molecular species were quantified using specific precursor product ion transitions. Calibration was accomplished by reference to a set of synthetic fatty acid AMPP derivatives prepared from accurate mass standards obtained from NuCheck Prep. Product ion spectra were generated for the most abundant species to determine the position of double bonds.

2.7 Statistical analysis

Data are expressed as mean ± SE. Results were analyzed by one or two-way ANOVA followed by Tukey's or Bonferroni's post-test, respectively. Values of P<0.05 were considered statistically significant.

3. Results

3.1 GV sPLA2-modified LDL promote TNF-α and IL-6 secretion by J-774 macrophages

In previous studies we determined that human LDL hydrolyzed by Group V sPLA2 induces macrophage foam cell formation to a significantly greater extent than native LDL [10]. In this study we investigated whether GV-LDL has other pro-atherosclerotic effects on macrophages in addition to intracellular lipid accumulation. J-774 macrophage-like cells were incubated with 0.1mg/ml GV-LDL or control-LDL, and then cells and media were collected at selected intervals to quantify the expression of inflammatory cytokines. Incubations with GV-LDL led to a significant, time-dependent increase in both TNF-α and IL-6 mRNA expression compared to baseline values, whereas control-LDL lacked this effect (Fig. 1A, B). Accompanying these increases in mRNA expression were significant ∼11- and 6.5-fold increases in the amount of TNF-α and IL-6, respectively, in media from macrophages incubated with GV-LDL for 8 h compared to control LDL (Fig. 1 C, D). In contrast, addition of equivalent amounts of GV sPLA2 in the absence of LDL did not significantly affect cytokine secretion (Fig. 1 C, D grey bars). Similar effects on TNF-α secretion also occurred when mouse peritoneal macrophages were incubated with GV-LDL (supplemental Fig. 1).

An external file that holds a picture, illustration, etc.
Object name is nihms179352f1.jpg
GV-LDL promotes TNF-alpha and IL-6 secretion from J-774 cells

J-774 cells were incubated with 100 μg/ml control-LDL, GV-LDL, or the equivalent amount of GV sPLA2 present in 100 μg/ml GV-LDL for the indicated times. (A, B) RNA was isolated from cells and TNF-α and IL-6 mRNA expression was quantified by qRT-PCR and normalized to 18S. Results are expressed as fold increase compared to baseline levels. (C, D) TNF-α and IL-6 protein in the media were quantified by ELISA using commercially available kits. Data are presented as mean±SE and are representative of 3 independent experiments. * indicates p<0.05 and ** indicates p<0.01 compared to control-LDL.

3.2 GV-LDL induces nuclear translocation of NFκB

NFκB is a transcription factor mediating the inflammatory responses triggered by various stimuli. A required step for NFκB activation is its transport to the nucleus, which occurs subsequent to the phosphorylation and ubiquitination of the inhibitor of NFκB, IκB. To investigate whether GV-LDL induces cytokine expression through NFκB activation, the intracellular localization of the NFκB p65 subunit was assessed in J-774 cells after 1 h treatment with either control-LDL or GV-LDL. For comparison, J-774 cells were treated with 100 ng/ml LPS. As expected, analysis by confocal microscopy revealed that the bulk of p65 is present in the nucleus of J-774 cells after treatment with LPS, consistent with activation of NFκB (Fig. 2A, upper left panel, arrows). Interestingly, cells treated with GV-LDL also showed nuclear localization of p65 (Fig. 2A, upper right). In contrast, there was no evidence for translocation of p65 to the nucleus of cells treated with control-LDL (Fig. 2A, lower left). The use of a non-specific primary antibody confirmed the specificity of the immunofluorescence in these experiments (Fig. 2A, lower right). Quantification of nuclear p65-positive fluorescence (expressed as the percent of total cellular fluorescence) confirmed nuclear translocation of NFκB in cells treated with either LPS or GV-LDL, but not control-LDL (Fig. 2B).

An external file that holds a picture, illustration, etc.
Object name is nihms179352f2.jpg
GV-LDL induces NFκB nuclear translocation

(A) J-774 cells were incubated with 100 ng/ml LPS, 100 μg/ml GV-LDL or 100 μg/ml control-LDL for 1h and then fixed in 10% formaldehyde. NFκB was visualized by indirect immunofluorescence using mouse anti-p65 IgG as primary antibody (green). The lower right panel shows cells immunostained with a non-specific primary IgG. Cell cytoskeleton was visualized with rhodamine phalloidin (red) and nuclei with DAPI (blue). Arrows indicate nuclear localization of p65. (B) Nuclear NFκB was quantified in 50 randomly selected cells per group and expressed as the amount of p65-positive fluorescence associated with the nucleus as a percent of total cellular fluorescence. ** indicates p<0.01 compared to control-LDL, # indicates p<0.01 compared to GV-LDL.

3.3 NFκB inhibition reduces cytokine secretion induced by GV-LDL

To provide additional evidence that GV-LDL induces cytokine secretion through activation of NFκB, we incubated cells with GV-LDL in the presence of two different NFκB inhibitors: SN50, a cell-permeable inhibitory peptide of NFκB translocation [29], or 6-amino-4-phenethylaminoquinazoline (quinazoline), an inhibitor of NFκB transcriptional activation [24]. Cells were pre-incubated for 1 h with SN50 or control peptide, or quinazoline or vehicle, followed by 8 h incubations with 0.1 mg/ml control-LDL or GV-LDL in the presence of the same treatment (Fig. 3). Whereas inhibition of NFκB activation had no effect on the amount of TNF-α secreted by cells incubated with control-LDL, the increase in TNF-α induced by GV-LDL was significantly blocked in cells incubated with either SN50 (Fig. 3A) or quinazoline (Fig. 3B).

An external file that holds a picture, illustration, etc.
Object name is nihms179352f3.jpg
The induction of inflammatory cytokines by GV-LDL is dependent on NFκB activation

(A) J-774 cells were pre-incubated for 1 h with 20 μM control peptide or the NFκB specific inhibitor SN50. Cells were then incubated for 8h with 100 μg/ml control-LDL or GV-LDL in the presence of the same peptide, and the amount of TNF-α in the media was quantified. (B) J-774 cells were pre-incubated for 1 h with 10 μM 6-amino-4-phenethylaminoquinazoline (quinazoline) or vehicle, and then incubated under the same treatment conditions for 8h with 100 μg/ml control-LDL or GV-LDL. Data are presented as mean±SE of triplicate determinations and are representative of 3 independent experiments. ** indicates p<0.01 compared to cells incubated in the presence of inhibitor.

3.4 Albumin-bound lipolytic products present in GV-LDL promote cytokine secretion by J-774 cells

It was of interest to determine whether GV-LDL leads to NFκB activation through actions of the structurally altered LDL particle or, alternatively, lipid products generated from the hydrolysis of LDL-PL. In our studies, LDL was hydrolyzed in the presence of physiologic albumin concentrations (2%). Under these conditions, FFA liberated by sPLA2 are transferred from LDL to albumin [23, 30]. We therefore considered density gradient ultracentrifugation a reasonable method to effectively separate the hydrolyzed LDL particle from its lipolytic products (Supplemental Fig. 2). We tested the ability of re-isolated LDL (top fractions after density-gradient centrifugation) and the albumin/FFA-enriched fraction (bottom fractions after density-gradient centrifugation) to induce cytokine release in J-774 cells. In order for responses to be directly compared, cells were incubated with either 0.1 mg/ml of the re-floated LDL or 2.0 mg/ml of the albumin-containing fraction, concentrations corresponding to those used prior to density-gradient ultracentrifugation (see Fig. 1). Although the refloated GV-LDL was somewhat more effective in stimulating cytokine release compared to the refloated control-LDL, this fraction was clearly less potent compared to the albumin fraction derived from GV-LDL or a mixture of the bottom and top fractions derived from GV-LDL (Fig. 4A,C). TNF-α and IL-6 secretion induced by the albumin-enriched fraction was significantly inhibited by quinazoline, suggesting a role for NFκB in the pro-inflammatory effect (Fig. 4 B,D).

An external file that holds a picture, illustration, etc.
Object name is nihms179352f4.jpg
The albumin-bound lipolytic products promote cytokine secretion by J-774 cells

J-774 cells were incubated for 8h with 100 μg/ml re-floated control-LDL or GV-LDL (top), or 2 mg/ml albumin-enriched fractions derived from control-LDL or GV-LDL (bottom), or a mixture of the two fractions (recombined), and the amount of (A) TNF-α and (C) IL-6 protein present in the media was quantified by ELISA. * indicates p<0.05 and ** indicates p<0.01 compared to the corresponding fraction from control-LDL. (B, D) J-774 cells were incubated for 8 h with the bottom fraction derived from GV-LDL (100 μg/ml) in the presence of vehicle or 10 μM quinazoline. * indicates p<0.05 compared to vehicle. Data are presented as mean±SE and are representative of at least 3 experiments.

3.5 Albumin-bound 18:1 present in GV-LDL promote cytokine secretion by J-774 cells

Our data indicate that lipolytic products present in GV-LDL promote macrophage inflammatory responses. Thus, it was of interest to identify the FFA and lyso-PC species that are generated when LDL is hydrolyzed by GV sPLA2. Lipid extracts from control-LDL and GV-LDL were analyzed by HPLC ESI tandem Mass spectrometry as described in Experimental Procedures. The newly formed FFA and lyso-PC species (defined as the molecular species present on re-isolated GV-LDL or the albumin-enriched fraction derived from GV-LDL after subtracting the constitutively present species on the corresponding fractions from control-LDL) are shown in Fig. 5 A and B. The most abundant newly formed FFA species were 18:2 (linoleic acid) and 18:1 (oleic acid, Fig. 5A). As expected, most of the FFA was associated with the albumin fraction. The most abundant newly formed lyso-PC species contained 14:0 (myristic), 16:1 (palmitic), and 18:2 fatty acyl groups. The MS/MS analysis identified 18:1 as delta 9 and 18:2 as delta 9 delta 12. Interestingly, lyso-PC species were approximately equally distributed between LDL particles and albumin (Fig. 5B).

An external file that holds a picture, illustration, etc.
Object name is nihms179352f5.jpg
Analysis of FFA and lyso-PC generated after hydrolysis of LDL

(A, B) Lipids were extracted from 100 μl aliquots of the top (LDL) and bottom (BSA) fractions derived from control-LDL and GV-LDL after ultracentrifugation and analyzed for molecular species of FFA and lyso-PL by HPLC ESI tandem Mass spectrometry. Newly formed FFA and lyso-PC are defined as the molecular species present on re-isolated GV-LDL or the albumin-enriched fraction derived from GV-LDL after subtracting the species present on the corresponding fractions from control-LDL. (C,D) J-774 cells were incubated for 8h with 100 μM of albumin-bound 18:1 or 18:2 and the amount of TNF-α and IL-6 secreted into the media was quantified by ELISA. Data are averaged from 2 separate experiments, each performed in triplicate. * indicates p<0.05, ** indicates p<0.01.

To investigate whether either 18:1 or 18:2 comprise the bioactive component of GV-LDL, J-774 cells were incubated with 100 μM albumin-conjugated 18:1 or 18:2, concentration comparable to the amount of the FFA present in the GV-LDL mix. Incubations with 18:1 resulted in significantly increased TNF-α and IL-6 secretion (Fig. 5C, D). Although 18:2 resulted in modestly increased TNF-α and IL-6 secretion, this effect was not significant.

3.1 GV sPLA2-modified LDL promote TNF-α and IL-6 secretion by J-774 macrophages

In previous studies we determined that human LDL hydrolyzed by Group V sPLA2 induces macrophage foam cell formation to a significantly greater extent than native LDL [10]. In this study we investigated whether GV-LDL has other pro-atherosclerotic effects on macrophages in addition to intracellular lipid accumulation. J-774 macrophage-like cells were incubated with 0.1mg/ml GV-LDL or control-LDL, and then cells and media were collected at selected intervals to quantify the expression of inflammatory cytokines. Incubations with GV-LDL led to a significant, time-dependent increase in both TNF-α and IL-6 mRNA expression compared to baseline values, whereas control-LDL lacked this effect (Fig. 1A, B). Accompanying these increases in mRNA expression were significant ∼11- and 6.5-fold increases in the amount of TNF-α and IL-6, respectively, in media from macrophages incubated with GV-LDL for 8 h compared to control LDL (Fig. 1 C, D). In contrast, addition of equivalent amounts of GV sPLA2 in the absence of LDL did not significantly affect cytokine secretion (Fig. 1 C, D grey bars). Similar effects on TNF-α secretion also occurred when mouse peritoneal macrophages were incubated with GV-LDL (supplemental Fig. 1).

An external file that holds a picture, illustration, etc.
Object name is nihms179352f1.jpg
GV-LDL promotes TNF-alpha and IL-6 secretion from J-774 cells

J-774 cells were incubated with 100 μg/ml control-LDL, GV-LDL, or the equivalent amount of GV sPLA2 present in 100 μg/ml GV-LDL for the indicated times. (A, B) RNA was isolated from cells and TNF-α and IL-6 mRNA expression was quantified by qRT-PCR and normalized to 18S. Results are expressed as fold increase compared to baseline levels. (C, D) TNF-α and IL-6 protein in the media were quantified by ELISA using commercially available kits. Data are presented as mean±SE and are representative of 3 independent experiments. * indicates p<0.05 and ** indicates p<0.01 compared to control-LDL.

3.2 GV-LDL induces nuclear translocation of NFκB

NFκB is a transcription factor mediating the inflammatory responses triggered by various stimuli. A required step for NFκB activation is its transport to the nucleus, which occurs subsequent to the phosphorylation and ubiquitination of the inhibitor of NFκB, IκB. To investigate whether GV-LDL induces cytokine expression through NFκB activation, the intracellular localization of the NFκB p65 subunit was assessed in J-774 cells after 1 h treatment with either control-LDL or GV-LDL. For comparison, J-774 cells were treated with 100 ng/ml LPS. As expected, analysis by confocal microscopy revealed that the bulk of p65 is present in the nucleus of J-774 cells after treatment with LPS, consistent with activation of NFκB (Fig. 2A, upper left panel, arrows). Interestingly, cells treated with GV-LDL also showed nuclear localization of p65 (Fig. 2A, upper right). In contrast, there was no evidence for translocation of p65 to the nucleus of cells treated with control-LDL (Fig. 2A, lower left). The use of a non-specific primary antibody confirmed the specificity of the immunofluorescence in these experiments (Fig. 2A, lower right). Quantification of nuclear p65-positive fluorescence (expressed as the percent of total cellular fluorescence) confirmed nuclear translocation of NFκB in cells treated with either LPS or GV-LDL, but not control-LDL (Fig. 2B).

An external file that holds a picture, illustration, etc.
Object name is nihms179352f2.jpg
GV-LDL induces NFκB nuclear translocation

(A) J-774 cells were incubated with 100 ng/ml LPS, 100 μg/ml GV-LDL or 100 μg/ml control-LDL for 1h and then fixed in 10% formaldehyde. NFκB was visualized by indirect immunofluorescence using mouse anti-p65 IgG as primary antibody (green). The lower right panel shows cells immunostained with a non-specific primary IgG. Cell cytoskeleton was visualized with rhodamine phalloidin (red) and nuclei with DAPI (blue). Arrows indicate nuclear localization of p65. (B) Nuclear NFκB was quantified in 50 randomly selected cells per group and expressed as the amount of p65-positive fluorescence associated with the nucleus as a percent of total cellular fluorescence. ** indicates p<0.01 compared to control-LDL, # indicates p<0.01 compared to GV-LDL.

3.3 NFκB inhibition reduces cytokine secretion induced by GV-LDL

To provide additional evidence that GV-LDL induces cytokine secretion through activation of NFκB, we incubated cells with GV-LDL in the presence of two different NFκB inhibitors: SN50, a cell-permeable inhibitory peptide of NFκB translocation [29], or 6-amino-4-phenethylaminoquinazoline (quinazoline), an inhibitor of NFκB transcriptional activation [24]. Cells were pre-incubated for 1 h with SN50 or control peptide, or quinazoline or vehicle, followed by 8 h incubations with 0.1 mg/ml control-LDL or GV-LDL in the presence of the same treatment (Fig. 3). Whereas inhibition of NFκB activation had no effect on the amount of TNF-α secreted by cells incubated with control-LDL, the increase in TNF-α induced by GV-LDL was significantly blocked in cells incubated with either SN50 (Fig. 3A) or quinazoline (Fig. 3B).

An external file that holds a picture, illustration, etc.
Object name is nihms179352f3.jpg
The induction of inflammatory cytokines by GV-LDL is dependent on NFκB activation

(A) J-774 cells were pre-incubated for 1 h with 20 μM control peptide or the NFκB specific inhibitor SN50. Cells were then incubated for 8h with 100 μg/ml control-LDL or GV-LDL in the presence of the same peptide, and the amount of TNF-α in the media was quantified. (B) J-774 cells were pre-incubated for 1 h with 10 μM 6-amino-4-phenethylaminoquinazoline (quinazoline) or vehicle, and then incubated under the same treatment conditions for 8h with 100 μg/ml control-LDL or GV-LDL. Data are presented as mean±SE of triplicate determinations and are representative of 3 independent experiments. ** indicates p<0.01 compared to cells incubated in the presence of inhibitor.

3.4 Albumin-bound lipolytic products present in GV-LDL promote cytokine secretion by J-774 cells

It was of interest to determine whether GV-LDL leads to NFκB activation through actions of the structurally altered LDL particle or, alternatively, lipid products generated from the hydrolysis of LDL-PL. In our studies, LDL was hydrolyzed in the presence of physiologic albumin concentrations (2%). Under these conditions, FFA liberated by sPLA2 are transferred from LDL to albumin [23, 30]. We therefore considered density gradient ultracentrifugation a reasonable method to effectively separate the hydrolyzed LDL particle from its lipolytic products (Supplemental Fig. 2). We tested the ability of re-isolated LDL (top fractions after density-gradient centrifugation) and the albumin/FFA-enriched fraction (bottom fractions after density-gradient centrifugation) to induce cytokine release in J-774 cells. In order for responses to be directly compared, cells were incubated with either 0.1 mg/ml of the re-floated LDL or 2.0 mg/ml of the albumin-containing fraction, concentrations corresponding to those used prior to density-gradient ultracentrifugation (see Fig. 1). Although the refloated GV-LDL was somewhat more effective in stimulating cytokine release compared to the refloated control-LDL, this fraction was clearly less potent compared to the albumin fraction derived from GV-LDL or a mixture of the bottom and top fractions derived from GV-LDL (Fig. 4A,C). TNF-α and IL-6 secretion induced by the albumin-enriched fraction was significantly inhibited by quinazoline, suggesting a role for NFκB in the pro-inflammatory effect (Fig. 4 B,D).

An external file that holds a picture, illustration, etc.
Object name is nihms179352f4.jpg
The albumin-bound lipolytic products promote cytokine secretion by J-774 cells

J-774 cells were incubated for 8h with 100 μg/ml re-floated control-LDL or GV-LDL (top), or 2 mg/ml albumin-enriched fractions derived from control-LDL or GV-LDL (bottom), or a mixture of the two fractions (recombined), and the amount of (A) TNF-α and (C) IL-6 protein present in the media was quantified by ELISA. * indicates p<0.05 and ** indicates p<0.01 compared to the corresponding fraction from control-LDL. (B, D) J-774 cells were incubated for 8 h with the bottom fraction derived from GV-LDL (100 μg/ml) in the presence of vehicle or 10 μM quinazoline. * indicates p<0.05 compared to vehicle. Data are presented as mean±SE and are representative of at least 3 experiments.

3.5 Albumin-bound 18:1 present in GV-LDL promote cytokine secretion by J-774 cells

Our data indicate that lipolytic products present in GV-LDL promote macrophage inflammatory responses. Thus, it was of interest to identify the FFA and lyso-PC species that are generated when LDL is hydrolyzed by GV sPLA2. Lipid extracts from control-LDL and GV-LDL were analyzed by HPLC ESI tandem Mass spectrometry as described in Experimental Procedures. The newly formed FFA and lyso-PC species (defined as the molecular species present on re-isolated GV-LDL or the albumin-enriched fraction derived from GV-LDL after subtracting the constitutively present species on the corresponding fractions from control-LDL) are shown in Fig. 5 A and B. The most abundant newly formed FFA species were 18:2 (linoleic acid) and 18:1 (oleic acid, Fig. 5A). As expected, most of the FFA was associated with the albumin fraction. The most abundant newly formed lyso-PC species contained 14:0 (myristic), 16:1 (palmitic), and 18:2 fatty acyl groups. The MS/MS analysis identified 18:1 as delta 9 and 18:2 as delta 9 delta 12. Interestingly, lyso-PC species were approximately equally distributed between LDL particles and albumin (Fig. 5B).

An external file that holds a picture, illustration, etc.
Object name is nihms179352f5.jpg
Analysis of FFA and lyso-PC generated after hydrolysis of LDL

(A, B) Lipids were extracted from 100 μl aliquots of the top (LDL) and bottom (BSA) fractions derived from control-LDL and GV-LDL after ultracentrifugation and analyzed for molecular species of FFA and lyso-PL by HPLC ESI tandem Mass spectrometry. Newly formed FFA and lyso-PC are defined as the molecular species present on re-isolated GV-LDL or the albumin-enriched fraction derived from GV-LDL after subtracting the species present on the corresponding fractions from control-LDL. (C,D) J-774 cells were incubated for 8h with 100 μM of albumin-bound 18:1 or 18:2 and the amount of TNF-α and IL-6 secreted into the media was quantified by ELISA. Data are averaged from 2 separate experiments, each performed in triplicate. * indicates p<0.05, ** indicates p<0.01.

To investigate whether either 18:1 or 18:2 comprise the bioactive component of GV-LDL, J-774 cells were incubated with 100 μM albumin-conjugated 18:1 or 18:2, concentration comparable to the amount of the FFA present in the GV-LDL mix. Incubations with 18:1 resulted in significantly increased TNF-α and IL-6 secretion (Fig. 5C, D). Although 18:2 resulted in modestly increased TNF-α and IL-6 secretion, this effect was not significant.

4. Discussion

Several lines of evidence suggest that GV sPLA2 modification of LDL results in structural alterations of the lipoprotein particle that promote atherosclerotic lipid accumulation [8-10, 31-32]. In this study we provide novel evidence that products generated through LDL hydrolysis activate NFκB, a transcription factor and key regulator of inflammation that has been implicated in atherosclerotic processes [33]. Our data show that incubations with GV-LDL, but not control LDL, result in nuclear translocation of the p65 subunit of NFκB and increased expression of NFκB target genes, namely TNF-α and IL-6. Importantly, SN50, a cell-permeable peptide that inhibits nuclear translocation of the transcription factor active complex [29], and 6-amino-4-phenetylaminoquinazoline, an inhibitor of NFκB transcriptional activation [24], effectively blocked TNF-α and IL-6 upregulation by GV-LDL. Analysis of data from all of our studies using the cell-permeable peptides suggested these reagents activate macrophages to some extent. Nevertheless, the effect of GV-LDL on macrophage activation was clearly blunted when nuclear translocation of NFκB was inhibited. In addition, treatments with quinazoline, an inhibitor of NFκB transcriptional activation, also blocked the pro-inflammatory effect of GV-LDL without elevating baseline cytokine secretion. The conclusion that inflammatory cytokine induction was due to a specific product of LDL hydrolysis and not an experimental artifact such as endotoxin contamination was substantiated by control experiments in which preparations of LDL (“control-LDL”) were subjected to identical manipulations as GV-LDL, except that GV sPLA2 was omitted from hydrolysis reactions. We also verified that all reagents besides LPS used in this study were endotoxin-free, and that incubating cells with equivalent amounts of GV sPLA2 by itself did not evoke NFκB activation in macrophages.

After density gradient ultracentrifugation, the bioactive component of GV-LDL was recovered in a FFA-enriched albumin fraction and not the re-floated GV-LDL. Thus, the effect of GV-LDL on NFκB-mediated inflammatory responses does not appear to be dependent on binding/uptake of the modified LDL particle, but is rather mediated by bioactive lipids generated through LDL hydrolysis. Analysis by HPLC ESI tandem mass spectrometry demonstrated that considerable amounts of both 18:1 and 18:2 were generated when LDL was hydrolyzed by GV sPLA2, confirming previously published studies [34]. We further defined these FFA species as 18:1 delta 9 and 18:2 delta 9 delta 12. Although newly formed lyso-PC species were also present on GV-LDL (containing primarily 14:0, 16:1, and 18:2), it seems unlikely that these represent the bioactive component since they partitioned approximately equally between the FFA-enriched albumin fraction and the re-floated GV-LDL. Notably, additional studies showed that treatments with 18:1 alone at concentrations approximating the amount present in unfractionated GV-LDL reproduced the effect of GV-LDL. Interestingly, evidence suggests that dietary monounsaturated fatty acids lead to cholesteryl oleate enrichment of LDL, which has been implicated in atherosclerotic processes in both experimental animals and in humans (recently reviewed in [35]). Our studies would suggest that oleate-enrichment of LDL phospholipids may also be detrimental. Other studies have reported pro-inflammatory activity of non-saturated fatty acids, namely 18:2, on endothelial cells [36]. These authors pointed to a pathway for NFκB activation involving phosphatidylinositol 3-kinase/amino kinase terminal (PI3K/Akt), extracellular signal regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK).

Several NFκB activation pathways have been identified in cells, with LPS being one of the most potent activators of both classic and alternative pathways (reviewed in [33]). Hence, LPS has been widely used as a stimulatory agent to study the effects of various molecules, including GV sPLA2 on inflammation. Using an siRNA approach, Ruiperez et al. showed that endogenous GV sPLA2 potentiates the effect of LPS on COX-2 activation in P388D1 macrophages [37]. In contrast, our studies were carried out in the absence of any other pro-inflammatory stimuli. While the amount of cytokine secretion induced by GV-LDL is relatively modest compared to maximal responses produced by LPS, the co-localization of GV sPLA2 and high concentrations of LDL in the vessel wall could provide an important mechanism for initiating macrophage activation.

A previous study by Karabina et al. investigated the effect of incubating human endothelial cells with LDL particles hydrolyzed by GX sPLA2 [38]. According to this report, GX sPLA2 hydrolysis results in the formation of an altered LDL particle that is capable of activating the MAP kinase pathway in endothelial cells, which leads to increased arachidonic acid release and increased expression of major adhesion molecules, including ICAM-1 and VCAM-1. Although re-isolation of GX-LDL by ultracentrifugation did not attenuate its ability to induce AA release, the authors did not rule out the possibility that hydrolytic products generated through the action of GX sPLA2 act alone or in combination with the modified LDL to induce biological responses. Whether lipolytic products were effectively removed from the re-isolated GX-LDL was not directly determined. Since hydrolysis reactions were carried out in the presence of sub-physiological concentrations of BSA (0.0125%), it seems likely that under the conditions of the study potentially bioactive lipids liberated by sPLA2 did indeed accumulate in the hydrolyzed LDL particle, as previously described by Kleinman et al. [30].

According to a previously published report, human recombinant GIB and GX sPLA2 induce TNF-α and IL-6 secretion from human alveolar macrophages through a mechanism that is independent of their phospholipase activity [39]. These findings contrast to the current study, where incubations with GV sPLA2 in the absence of LDL failed to evoke cytokine secretion. Differences in cell types and the amount of sPLA2 used in the two studies likely account for the discrepant results. The authors of the previous study speculated that sPLA2-induced cytokine production may be mediated through high affinity binding to the M-type sPLA2 receptor, which is highly expressed in human alveolar macrophages. Based on results from RT-PCR analysis, J-774 cells express little/no M-type receptor mRNA (our unpublished data). Furthermore, the effect of GIB and GX on cytokine secretion did not become statistically significant until relatively high concentrations were used (1μg/ml). We estimate based on silver staining of partially purified enzyme that J-774 cells were exposed to <1 ng/ml GV sPLA2 in the current study.

Our finding that GV sPLA2 hydrolysis of LDL leads to increased pro-inflammatory responses through NFκB activation differs from lipoprotein lipase action on triglyceride-rich lipoproteins, which appears to effectively limit inflammatory responses through the production of ligands for PPARs [40-41]. Although sPLA2's can generate lipid mediators (FFAs, prostaglandins) that are potential PPAR ligands, evidence that this occurs is controversial [40, 42-43]. In the case of lipoprotein-associated phospholipase A2 (Lp-PLA2) the enzyme effectively hydrolyzes truncated acyl chains present on oxidatively modified LDL to release lyso-phosphatidylcholine and oxidized FFAs [44]. Thus, different FFA-generating lipases may induce distinct responses s generated. Our finding that hydrolytic products, likely 18:1, liberated from LDL by GV sPLA2 induce macrophages to secrete inflammatory cytokines may have important implications for innate immunity and chronic inflammation in atherosclerosis.

Supplementary Material

01

02

03

01

Click here to view.(26K, doc)

02

Click here to view.(127K, ppt)

03

Click here to view.(147K, ppt)

Acknowledgments

This work was supported by National Institutes of Health grant P01 HL080100 (to NRW). This material is the result of work supported with resources and the use of facilities at the Lexington VAMC. HPLC ESI tandem Mass spectrometry is supported by NIHGM50388-18 and NCRRRP20021594 (to AJM).

We thank Kathy Forrest for her technical assistance, Dr. David Watt for the FFA derivatization reagents and Richard Charnigo for his help with statistical analyses.

Department of Internal Medicine Endocrinology Division, University of Kentucky, Lexington, KY 40536
Graduate Center for Nutritional Sciences, University of Kentucky, Lexington, KY 40536
Cardiovascular Research Center, University of Kentucky, Lexington, KY 40536
Veterans Affairs Medical Center, Lexington, KY 40536
Address correspondence to: Boris Boyanovsky, Department of Internal Medicine, University of Kentucky Medical Center, 533 CT Wethington Bldg., 900 S. Limestone St., Lexington, KY 40536-0200, Tel: (859) 323-4933 x 81383; Fax: (859) 257-3646; ude.yku@2ayobbb
Publisher's Disclaimer

Abstract

Objective

Previous studies have established that hydrolysis of LDL by Group V secretory phospholipase A2 (GV sPLA2) generates a modified particle capable of inducing macrophage foam cell formation. The aim of the present study was to determine whether GV sPLA2-hydrolyzed LDL (GV-LDL) produces pro-atherogenic effects in macrophages independent of cholesterol accumulation.

Methods and Results

J-774 cells incubated with GV-LDL produced more TNF-α and IL-6 compared to cells incubated with control-LDL. Indirect immunofluorescence showed that GV-LDL but not control-LDL induced nuclear translocation of NFκB. Inhibitors of NFκB activation, effectively blocked cytokine production induced by GV-LDL. Control-LDL and GV-LDL were separated from albumin present in reaction mixtures by ultracentrifugation. The albumin fraction derived from GV-LDL contained 80% of the FFA generated and was more potent than the re-isolated GV-LDL in inducing pro-inflammatory cytokine secretion. Linoleic acid (18:2) and oleic acid (18:1) were the most abundant FFAs generated, whereas newly formed lyso-PCs contained 14:0 (myristic), 16:1 (palmitic), and 18:2 fatty acyl groups. Experiments with synthetic FFA showed that 18:1 induced J-774 cells to secrete TNF-α and IL-6.

Conclusions

These results indicate that in addition to promoting atherosclerotic lipid accumulation in macrophages, GV sPLA2 hydrolysis of LDL leads to activation of NFκB, a key regulator of inflammation.

Keywords: atherosclerosis, inflammation, lipoprotein modification, tumor necrosis factor-alpha, interleukin-6, NFκB
Abstract

Footnotes

Authors have no conflicts of interest to disclose.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Footnotes

References

  • 1. Williams KJ, Tabas IThe response-to-retention hypothesis of early atherogenesis. Arterioscler Thromb Vasc Biol. 1995;15:551–61.[Google Scholar]
  • 2. Libby PInflammation in atherosclerosis. Nature. 2002;420:868–74.[PubMed][Google Scholar]
  • 3. Lambeau G, Gelb MHBiochemistry and physiology of mammalian secreted phospholipases A2. Annu Rev Biochem. 2008;77:495–520.[PubMed][Google Scholar]
  • 4. Boyanovsky BB, Webb NRBiology of secretory phospholipase A2. Cardiovasc Drugs Ther. 2009;23:61–72.[Google Scholar]
  • 5. Jönsson-Rylander AC, Lundin S, Rosengren B, Pettersson C, Hurt-Camejo ERole of secretory phospholipases in atherogenesis. Curr Atheroscler Rep. 2008;10:252–9.[PubMed][Google Scholar]
  • 6. Murakami M, Kudo INew phospholipase A(2) isozymes with a potential role in atherosclerosis. Curr Opin Lipidol. 2003;14:431–6.[PubMed][Google Scholar]
  • 7. Webb NRSecretory phospholipase A2 enzymes in atherogenesis. Curr Opin Lipidol. 2005;16:341–4.[PubMed][Google Scholar]
  • 8. Kimura-Matsumoto M, Ishikawa Y, Komiyama K, Tsuruta T, Murakami M, Masuda S, et al Expression of secretory phospholipase A2s in human atherosclerosis development. Atherosclerosis. 2008;196:81–91.[PubMed][Google Scholar]
  • 9. Hanasaki K, Yamada K, Yamamoto S, Ishimoto Y, Saiga A, Ono T, et al Potent modification of low density lipoprotein by group X secretory phospholipase A2 is linked to macrophage foam cell formation. J Biol Chem. 2002;277:29116–24.[PubMed][Google Scholar]
  • 10. Wooton-Kee CR, Boyanovsky B, Nasser M, de Villiers W, Webb NGroup V sPLA2 hydrolysis of low-density lipoprotein results in spontaneous particle aggregation and promotes macrophage foam cell formation. Arterioscler Thromb Vasc Biol. 2004;24:762–7.[PubMed][Google Scholar]
  • 11. Sato H, Kato R, Isogai Y, Saka G, Ohtsuki M, Taketomi Y, et al Analyses of group III secreted phospholipase A2 transgenic mice reveal potential participation of this enzyme in plasma lipoprotein modification, macrophage foam cell formation, and atherosclerosis. J Biol Chem. 2008;283:33483–97.[Google Scholar]
  • 12. Sartipy P, Camejo G, Svensson L, Hurt-Camejo EPhospholipase A2 modification of low density lipoproteins forms small high density particles with increased affinity for proteoglycans and glycosaminoglycans. J Biol Chem. 1999;274:25913–20.[PubMed][Google Scholar]
  • 13. Flood C, Gustafsson M, Pitas RE, Arnaboldi L, Walzem RL, Boren JMolecular mechanism for changes in proteoglycan binding on compositional changes of the core and the surface of low-density lipoprotein-containing human apolipoprotein B100. Arterioscler Thromb Vasc Biol. 2004;24:564–70.[PubMed][Google Scholar]
  • 14. Boyanovsky BB, Shridas P, Simons M, van der Westhuyzen DR, Webb NRSyndecan-4 mediates macrophage uptake of group V secretory phospholipase A2-modified LDL. J Lipid Res. 2009;50:641–50.[Google Scholar]
  • 15. Bostrom MA, Boyanovsky B, Jordan CT, Wadsworth MP, Taatjes DJ, de Beer FC, Webb NRGroup V secretory phospholipase A2 promotes atherosclerosis: evidence from genetically altered mice. Arterioscler Thromb Vasc Biol. 2007;27:600–6.[PubMed][Google Scholar]
  • 16. Boyanovsky B, Zack M, Forrest K, Webb NRThe capacity of group v sPLA2 to increase atherogenicity of apoE-/- and LDLR-/- mouse LDL in vitro predicts its atherogenic role in vivo. Arterioscler Thromb Vasc Biol. 2009;29:532–8.[Google Scholar]
  • 17. Curfs DMJ, Ghesquiere SAI, Vergouwe MN, van der Made I, Gijbels MJ, Greaves DR, Verbeek JS, Hofker MH, de Winther MPMacrophage secretory phospholipase A2 group X enhances anti-inflammatory responses, promotes lipid accumulation, and contributes to aberrant lung pathology. J Biol Chem. 2008;283:21640–21648.[PubMed][Google Scholar]
  • 18. Lee JY, Plakidas A, Lee WH, Heikkinen A, Chanmugam P, Bray G, Hwang DHDifferential modulation of Toll-like receptors by fatty acids: preferential inhibition by n-3 polyunsaturated fatty acids. J Lipid Res. 2003;44:479–86.[PubMed][Google Scholar]
  • 19. Lee JY, Sohn KH, Rhee SH, Hwang DSaturated fatty acids, but not unsaturated fatty acids, induce the expression of cyclooxygenase-2 mediated through toll-like receptor 4. J Biol Chem. 2001;276:16683–9.[PubMed][Google Scholar]
  • 20. Erridge C, Samani NJSaturated fatty acids do not directly stimulate toll-like receptor signaling. Arterioscler Thromb Vasc Biol. 2009;29:1944–9.[PubMed][Google Scholar]
  • 21. Zumwalt JW, Thunstrom BJ, Spangelo BLInterleukin-1β and catecholamines synergistically stimulate interleukin-6 release from rat C6 glioma cells in vitro: a potential role for lysophosphatidylcholine. Endocrinology. 1999;140:888–96.[PubMed][Google Scholar]
  • 22. Yan JJ, Jung JS, Lee JE, Lee J, Huh SO, Kim HS, Jung KC, Cho JY, Nam JS, Suh HW, Kim YH, Song DKTherapeutic effects of lysophosphatidylcholine in experimental sepsis. Nat Med. 2004;10:124–5.[PubMed][Google Scholar]
  • 23. Boyanovsky BB, van der Westhuyzen DR, Webb NRGroup V secretory phospholipase A2-modified low density lipoprotein promotes foam cell formation by a SR-A- and CD36-independent process that Involves cellular proteoglycans. J Biol Chem. 2005;280:32746–52.[PubMed][Google Scholar]
  • 24. Tobe M, Isobe Y, Tomizawa H, Nagasaki T, Takahashi H, Fukazawa T, et al Discovery of quinazolines as a novel structural class of potent inhibitors of NF-κB activation. Bioorg Med Chem. 2003;11:383–91.[PubMed][Google Scholar]
  • 25. Toborek M, Woo Lee Y, Kaiser S, Hennig BMeasurement of inflammatory properties of fatty acids in human endothelial cells. Methods Enzymol. 2002:198–219.[PubMed][Google Scholar]
  • 26. Su W, Yeku O, Olepu S, Genna A, Park JS, Ren H, et al 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), a phospholipase d pharmacological inhibitor that alters cell spreading and inhibits chemotaxis. Mol Pharmacol. 2009;75:437–46.[Google Scholar]
  • 27. Pamuklar Z, Federico L, Liu S, Umezu-Goto M, Dong A, Panchatcharam M, et al Autotaxin/lysopholipase d and lysophosphatidic acid regulate murine hemostasis and thrombosis. J Biol Chem. 2009;284:7385–94.[Google Scholar]
  • 28. Yang WC, Adamec J, Regnier FEEnhancement of the LC/MS analysis of fatty acids through derivatization and stable isotope coding. Anal Chem. 2007;79:5150–7.[PubMed][Google Scholar]
  • 29. Lin YZ, Yao S, Veach RA, Torgerson TR, Hawiger JInhibition of nuclear translocation of transcription factor NF-κB by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J Biol Chem. 1995;270:14255–8.[PubMed][Google Scholar]
  • 30. Kleinman Y, Krul E, Burnes M, Aronson W, Pfleger B, Schonfeld GLipolysis of LDL with phospholipase A2 alters the expression of selected apoB-100 epitopes and the interaction of LDL with cells. J Lipid Res. 1988;29:729–43.[PubMed][Google Scholar]
  • 31. Hurt-Camejo E, Camejo G, Sartipy PPhospholipase A2 and small, dense low-density lipoprotein. Curr Opin Lipidol. 2000;11:465–71.[PubMed][Google Scholar]
  • 32. Sartipy P, Camejo G, Svensson L, Hurt-Camejo EPhospholipase A2 modification of lipoproteins: potential effects on atherogenesis. Adv Exp Med Biol. 2002;507:3–7.[PubMed][Google Scholar]
  • 33. de Winther MP, Kanters E, Kraal G, Hofker MHNuclear Factor κB signaling in atherogenesis. Arterioscler Thromb Vasc Biol. 2005;25:904–14.[PubMed][Google Scholar]
  • 34. Gesquiere L, Cho W, Subbaiah PV. Role of group IIa and group V secretory phospholipases A(2) in the metabolism of lipoproteins. Substrate specificities of the enzymes and the regulation of their activities by sphingomyelin. Biochemistry. 2002;41:4911–20.[PubMed]
  • 35. Degirolamo C, Shelness GS, Rudel LLLDL cholesteryl oleate as a predictor for atherosclerosis: evidence from human and animal studies on dietary fat. J Lipid Res. 2009;50(Supplement):S434–9.[Google Scholar]
  • 36. Hennig B, Lei W, Arzuaga X, Ghosh DD, Saraswathi V, Toborek MLinoleic acid induces proinflammatory events in vascular endothelial cells via activation of PI3K/Akt and ERK1/2 signaling. J Nutr Biochem. 2006;17:766–72.[PubMed][Google Scholar]
  • 37. Ruiperez V, Casas J, Balboa MA, Balsinde JGroup V pPhospholipase A2-derived lysophosphatidylcholine mediates cyclooxygenase-2 induction in lipopolysaccharide-stimulated macrophages. J Immunol. 2007;179:631–8.[PubMed][Google Scholar]
  • 38. Karabina SA, Brocheriou I, Le Naour G, Agrapart M, Durand H, Gelb M, et al Atherogenic properties of LDL particles modified by human group X secreted phospholipase A2 on human endothelial cell function. FASEB J. 2006;20:2547–9.[PubMed][Google Scholar]
  • 39. Granata F, Petraroli A, Boilard E, Bezzine S, Bollinger J, Del Vecchio L, et al Activation of cytokine production by secreted phospholipase A2 in human lung macrophages expressing the M-type receptor. J Immunol. 2005;174:464–74.[PubMed][Google Scholar]
  • 40. Ziouzenkova O, Perrey S, Asatryan L, Hwang J, MacNaul KL, Moller DE, et al Lipolysis of triglyceride-rich lipoproteins generates PPAR ligands: Evidence for an antiinflammatory role for lipoprotein lipase. PNAS. 2003;100:2730–5.[Google Scholar]
  • 41. Ahmed W, Ziouzenkova O, Brown J, Devchand P, Francis SKM, Kanda T, et al PPARs and their metabolic modulation: new mechanisms for transcriptional regulation? J Intern Med. 2007;242:184–98.[PubMed][Google Scholar]
  • 42. Pawliczak R, Logun C, Madara P, Lawrence M, Woszczek G, Ptasinska A, et al Cytosolic phospholipase A2 group IVα but not secreted phospholipase A2 group IIa,V, or X induces interleukin-8 and cyclooxygenase-2 gene and protein expression through peroxisome proliferator-activated receptors γ 1 and 2 in human lung cells. J Biol Chem. 2004;279:48550–61.[PubMed][Google Scholar]
  • 43. Namgaladze D, Morbitzer D, von Knethen A, Brune BPhospholipase A2-modified low-density lipoprotein activates macrophage peroxisome proliferator-activated receptors. Arterioscler Thromb Vasc Biol. 2009:ATVBAHA.109.199232.[PubMed][Google Scholar]
  • 44. Zalewski A, Macphee CRole of lipoprotein-associated phospholipase A2 in atherosclerosis: biology, epidemiology, and possible therapeutic target. Arterioscler Thromb Vasc Biol. 2005;25:923–31.[PubMed][Google Scholar]
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.