©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Oxidized Low Density Lipoprotein Suppresses Activation of NFB in Macrophages via a Pertussis Toxin-sensitive Signaling Mechanism (*)

(Received for publication, December 6, 1994 )

Rodney E. Shackelford Uma K. Misra Kathryn Florine-Casteel Sheau-Fung Thai Salvatore V. Pizzo Dolph O. Adams(§)

From the Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The interaction of oxidized low density lipoprotein (ox-LDL) and macrophages is generally believed to be a significant inductive step in atherogenesis. Endocytosis of ox-LDL by scavenger receptors (SR) on macrophages is one result of this interaction, as is suppressed expression of several lipopolysaccharide (LPS)-stimulated, inflammatory genes such as tumor necrosis factor-alpha (TNF-alpha). Events subsequent to SR ligation, including intracellular signaling events if any, have not been established. We report here that ox-LDL initiates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate 2 (PIP(2)) and intracellular fluxes of Ca in macrophages, both of which are sensitive to pertussis toxin. ox-LDL also suppresses the LPS-induced binding of macrophage extracts to an NFkappaB sequence oligonucleotide and the LPS-initiated accumulation of RNA specific for TNF-alpha. These latter two effects are pertussis toxin-sensitive. Ligation of SR by ox-LDL thus initiates a pertussis toxin-sensitive signaling pathway in macrophages, which involves hydrolysis of PIP(2) and which can suppress expression of the TNF-alpha gene by modulating activation of NFkappaB.


INTRODUCTION

The interaction of ox-LDL (^1)with macrophages is generally believed to be an early and necessary event in initiating the atherosclerotic plaque(1) . Proteins or lipoproteins, chemically modified by a variety of reactions including oxidation, maleylation, and acetylation, are rapidly ingested by macrophages via a family of scavenger receptors (SR) originally described by Brown and Goldstein(2) . The two members of the family, which are expressed principally on macrophages and which have been cloned to date, are termed class A SRI and SRII and are elongated trimeric proteins, comprising six domains(3, 4) . ox-LDL induces pleiotropic changes in mononuclear phagocytes, including suppression of several, early genes induced by inflammatory stimuli such as bacterial lipopolysaccharide (LPS) or various cytokines(5, 6, 7, 8) . Most of these LPS-stimulated, early genes, such as TNF-alpha, have binding sites for NFkappaB in their promoter regions and in fact require these NFkappaB sites for transcription (9, 10, 11, 12, 13, 14, 15) . LPS initiates binding of dimers of the NFkappaB or Rel homology family including the NFkappaB(1)/Rel A (p50-p65) in extracts of macrophages and macrophage-like cell lines to oligonucleotides recognizing NFkappaB in electrophoretic mobility shift assays (EMSA) (16, 17, 18, 19) . Although the molecular events after ligation of SR by ox-LDL are not established(3) , ox-LDL does suppress LPS-stimulated expression of these genes in macrophages(5, 6, 7) .

We here report that ox-LDL initiates rapid increases in hydrolysis of PIP(2) and intracellular Ca, which are sensitive to pertussis toxin. Exposure of macrophages to ox-LDL suppresses LPS-induced activation/binding of NFkappaB to its cognate nucleotide and formation of a different retardation band in the EMSA; both of these effects of ox-LDL are sensitive to pertussis toxin. In the presence of ox-LDL plus pertussis toxin, LPS initiates binding of a p50-p65 dimer to an NFkappaB oligonucleotide and accumulation of mRNA specific for TNF-alpha.


EXPERIMENTAL PROCEDURES

Mice, Reagents, and Macrophages

Specific pathogen-free, inbred C57B/J mice (9-12 weeks of age) were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and housed in the Duke University Animal and Laboratory Isolation Facility in accordance with all institutional guidelines for care. Utmost precautions were taken such that the animals remained free from infection by environmental pathogens. LPS (prepared by Westphal phenolic extraction from Escherichia coli 026:136) and Brewer's thioglycolate broth were purchased from Difco. Bovine serum albumin was purchased from Sigma. RPMI 1640 without L-glutamine was purchased from Mediatech (Washington, D. C.). All reagents contained <0.125 ng of endotoxin (LPS)/ml as quantified by the Limulus amoebocyte lysate assay (Cape Cod Associates, Woods Hole, MA). Thioglycolate- and casein-elicited macrophages were obtained and cultured as reported previously(20) .

Isolation and Modification of LDL

LDL was isolated from human plasma according to published techniques(21) . In brief, freshly drawn human plasma was obtained from Duke University Medical Center Apheresis Center. EDTA and NaN(3) were added to 0.5 and 0.05%, respectively, to prevent oxidative modification and bacterial decomposition. The solvent density was adjusted to 1.020 by adding solid KBr. After ultracentrifugation for 20 h at 277,431 times g in a T1250 rotor (DuPont), the infranatant solvent density was adjusted to 1.060 with KBr. After a similar ultracentrifugation step, the supernatants (LDL) were collected, pooled, and dialyzed extensively at 4 °C against isotonic saline and 0.5 mM EDTA (pH 8.5). All lipoprotein preparations were tested for electrophoretic mobility, protein content (assay kit, Sigma), cholesterol (assay kit, Sigma), and endotoxin level (assay kit, Pyrotell, Woods Hole, MA).

Acetylation of LDL was carried out with acetic anhydride(22) . The degree of modification was assessed by determining reduced reactivity with trinitrobenzenesulfonic acid(23) . Oxidation of LDL was carried out by dialysis against isotonic saline free of EDTA, supplemented with 10 µM cupric sulfate or 10 µM ferrous sulfate for 4 days at 4 °C(22) . Oxidized preparations were dialyzed against 0.15 M NaCl with 0.5 mM EDTA (pH 8.5). The relative degree of oxidation was assayed by measurement of thiobarbituric acid-reactive substances(24) . For the lipoprotein preparations used in these experiments, thiobarbituric acid-reacting substances were less than 0.02 nmol/mg of LDL protein for native LDL and acetyl-LDL. For the various ox-LDL, thiobarbituric acid-reactive substances varied from 4.9 to 18.4 nmol/mg of LDL protein.

EMSA

Nuclear extracts were prepared by a modification of the procedure of Dignam et al.(25) . Each experimental treatment used 2.5 times 10^7 macrophages and was performed at 4 °C. Each tissue culture plate was then washed 2 times with 5 ml of phosphate-buffered saline. The cells were removed by scraping in 5 ml of phosphate-buffered saline and pelleted by centrifugation at 600 times g for 5 min and 4 °C. The cells were washed in 1 ml of Dignam solution A(25) , pelleted for 5 min, and suspended in 1.0 ml of solution A. The resulting cell suspensions were lysed with 20 strokes of an A-type pestle in a glass Dounce homogenizer (Wheaton, Millville, NJ). The nuclei were placed into a 5.0-ml ultracentrifuge tube (Sorvall), pelleted at 1,200 times g for 10 min in a swinging bucket rotor, and extracted in 0.05 ml of modified Dignam solution C (100 mM Hepes, pH 8.0, 25% glycerol, 1 mM leupeptin, and 400 mM NaCl). The final extracts were obtained after centrifugation at 25,000 times g for 7 min. This extract was then aliquoted into 1.5-ml Eppendorf tubes pushed halfway into crushed ice and stored at -70 °C. Protein concentrations were determined by the Bradford assay (26) using bovine serum albumin as the standard. DNA binding proteins present in nuclear extracts were analyzed using 3 µg of protein to bind the synthetic oligonucleotide (5`AAACAGGGGGCTTTCCCTCCTCAATATCAT3`)(27) . Each assay (0.02 ml) had a final concentration of 20,000 cpm of P-labeled DNA (approximately 0.1 ng), 1 µg of poly(dIbulletdC), 100 mM NaCl, 25 mM Hepes, 6.25% glycerol, 0.25 mM leupeptin (pH 8.0). The binding assays were loaded on 6% polyacrylamide gels (acrylamide:bisacrylamide, 29:1) in 0.25 times TBE buffer (22 mM Tris, 22 mM sodium borate, 0.5 mM EDTA, pH 8.0). After electrophoresis at 12 V/cm, the gels were dried and exposed to Kodak X-AR film. Oligonucleotides were labeled with [-P]ATP by T4 polynucleotide kinase and then annealed to the complementary DNA. Double-stranded DNAs were isolated by electrophoresis in 3% NuSieve agarose (Keene, NH) onto DEAE-cellulose membrane(28) . Data presented represent the results of at least three typical experiments.

Cellular Signaling

The methods employed in these studies have been described in detail elsewhere(29, 30) . In brief, measurement of [Ca](i) in murine peritoneal macrophages was quantified in peritoneal macrophages from C57Bl/6 mice. [Ca](i) measurements were performed using cells loaded with Fura-2/AM (Molecular Probes, Inc., Eugene, OR) and a digital imaging microscopy system. Ratio images were acquired at 30-s intervals. IP(3) was quantified as described(29, 30) . The effects of pertussis toxin (1 µg/ml, Sigma, preactivated with 40 mM dithiothreitol at 30 °C for 20 min) on IP(3) synthesis was studied employing [^3H]myoinositol-prelabeled murine peritoneal macrophages adherent to tissue culture plates. Thioglycolate-elicited peritoneal macrophages were harvested and adhered 2 h in inositol-free RPMI 1640 medium containing penicillin (12.5 units/ml), streptomycin (6.5 µg/ml), and 5% fetal calf serum and then labeled with [^3H]myoinositol (8 µCi/ml) for 16 h at 37 °C in the above medium. Monolayers were washed with Hanks' balanced salt solution containing 10 mM Hepes (pH 7.2) buffer containing 10 mM LiCl, 1 mM CaCl(2), and 1 mM MgCl(2), a volume of the buffer was added, and monolayers were preincubated for 3 min at 37 °C before stimulating with ox-LDL (15 µg/ml) for varying periods of time at 37 °C. The reaction was terminated by aspirating the medium and adding 6.25% HClO(4). The inositol phosphates were fractionated as described(29, 30) . To examine the effect of pertussis toxin on ox-LDL-stimulated IP(3) generation, labeled cells were treated with pertussis toxin (1 µg/ml) for 2 h at 37 °C and processed as above. Values are mean ± S.E.; similar results are obtained in duplicate experiments.

Northern Blot Preparations and Analogs

Northern blots were performed as described previously(20) . Films were scanned with a Molecular Dynamics PhosphorImager. To ensure that equivalent amounts of RNA were blotted to each lane the blots were rehybridized with the probe for -actin and the results normalized to actin. The data shown here typically represent the results of at least three experiments.


RESULTS

When extracts of LPS-stimulated macrophages were examined in an EMSA against the labeled NFkappaB-binding oligonucleotide, we found that they induced a distinct retardation band (Fig. 1A, band3 in lane6) comprising NFkappaB(1)/Rel A as determined by Western blot analysis (data not shown) as previously reported(16, 17, 18, 19) . In this assay, ox-LDL also induced a retardation band (Fig. 1A, band2, lane4), which was distinct from both the LPS-induced band and the constitutive band (Fig. 1A, band2, lane2) usually observed in macrophages or macrophage-like cell lines(16, 17, 18, 19, 20) . When macrophages were exposed concurrently to ox-LDL and LPS under conditions where ox-LDL suppressed the induction of inflammatory genes such as TNF-alpha by LPS(8) , the novel ox-LDL-induced band was observed, but the LPS-induced band and the constitutive band were significantly diminished or absent (Fig. 1A, lane8). By contrast, neither acetyl-LDL nor LDL induced a retardation band (Fig. 1B, lanes3 and 7). Neither acetyl-LDL, which also binds to SR, nor native LDL inhibited the LPS-initiated band (Fig. 1B, lanes5 and 6).


Figure 1: A, effects of ox-LDL on binding of NFkappaB. Lane1, free DNA; lane2, untreated; lane3, pertussis toxin; lane4, ox-LDL; lane5, ox-LDL + pertussis toxin; lane6, LPS; lane7, LPS + pertussis toxin; lane8, LPS + ox-LDL; lane9, LPS + ox-LDL + pertussis toxin. [ox-LDL], 100 µg/ml; [pertussis toxin], 1 µg/ml; [LPS], 10 ng/ml. Experimental details are given under ``Experimental Procedures''(15, 16, 17) . B, effects of acetylated and native LDL on the binding of NFkappaB. Lane1, free DNA; lane2, untreated; lane3, acetyl-LDL; lane4, LPS; lane5, LPS + acetyl-LDL; lane6, LPS + native LDL; lane7, native LDL. [acetyl LDL], 100 µg/ml; [native LDL], 100 µg/ml; [LPS], 10 ng/ml. Arrows indicate retardation bands 1, 2, and 3.



In light of proposed associations between the phosphoinositide signaling cascade and the activation of NFkappaB(30) , macrophages were exposed to ox-LDL and examined by digital imaging microscopy for fluxes in [Ca](i). Oxidized LDL, in fact, initiated rapid and transient increases in [Ca](i) in macrophages (Fig. 2A), though unmodified LDL did not. Images collected at 15-s intervals, rather than 30 s, usually revealed periodic calcium spiking upon stimulation with ox-LDL (data not shown). ox-LDL also stimulated increased hydrolysis of PIP(2), which was inhibitable by pretreatment of the macrophages with pertussis toxin (Fig. 2B). The increases in [Ca](i) were also sensitive to pertussis toxin (data not shown), suggesting a phospholipase coupled to a pertussis toxin-sensitive G protein is involved.


Figure 2: Effects of ox-LDL on cellular signaling. A, effects of ox-LDL on [Ca]. Arrow indicates addition of LDL (100 µg/ml concentration; ox-LDL also active at 15 µg/ml, data not shown). circle, macrophage + native LDL; bullet, macrophage + ox-LDL. Data points are averages of values obtained from three individually processed cells. B, effects of ox-LDL (15 µg/ml) and ox-LDL + pertussis toxin on formation of IP(3). Native LDL gave no increase in IP(3) (data not shown). (bullet, ox-LDL; circle, ox-LDL + pertussis toxin; x ± S.E.).



The effects of ox-LDL on binding of macrophage extracts to the NFkappaB nucleotide were also sensitive to pertussis toxin. The novel band in the EMSA assay, induced by ox-LDL, was reduced while the constitutive band was restored by prior treatment of the macrophages with pertussis toxin (Fig. 1A, compare lane5 with lane4). Inhibition of the LPS-induced band by ox-LDL was relieved by pertussis toxin (Fig. 1A, compare lane9 to lane8). When macrophages were stimulated by LPS, the enhanced levels of mRNA specific for TNF-alpha in Northern blots were inhibited by ox-LDL; this inhibition was relieved by pertussis toxin (Fig. 3).


Figure 3: Effects of pertussis toxin on expression of mRNA specific for TNF-alpha. Lane1, LPS; lane2, LPS + pertussis toxin; lane3, LPS + ox-LDL; lane4, LPS + ox-LDL + pertussis toxin. [LPS], 1 ng/ml; [ox-LDL], 100 µg/ml; [pertussis toxin], 1 µg/ml. Experimental details are given under ``Experimental Procedures''(19) .




DISCUSSION

The data presented here show that ox-LDL inhibits the LPS-induced binding of extracts of murine tissue macrophages to an NFkappaB oligonucleotide, which is found in the promoter of the murine TNF-alpha gene(9) . Previous studies in monocyte or macrophage-like cell lines have shown that extracts of unstimulated cells produce constitutive bands in an EMSA against NFkappaB sequences(16, 17, 18, 19) . The constitutive band generally comprises homodimers of (p50)(2) and (p52)(2). LPS, TNF-alpha, or phorbol 12-myristate 13-acetate can induce a band comprising a heterodimer of p50-p65, though LPS can also induce other bands over time(16) . The present data indicate that ox-LDL inhibits formation of the LPS-induced band and, concomitantly, initiates formation of a distinct and novel band. Studies of binding over 0-8 h indicate that the presence of the constitutive band wanes and waxes in parallel to waxing and waning of the ox-LDL-induced band (data not shown). This observation and binding of the novel band to the NFkappaB oligonucleotide raises the possibility that the ox-LDL-induced band comprises one or more NFkappaB subunits. If so, composition of the novel band could represent a number of possibilities, including combinations of an NFkappaB subunit with similar or additional NFkappaB subunits, IkappaB subunits, or other proteins such as bCl-3(19, 31) . Of interest, the ox-LDL-mediated suppression in vitro coincides in terms of dose, preincubation, and time with the ox-LDL-mediated suppression of four inflammatory genes by LPS(8) . The promoters of these four genes (i.e. TNF-alpha, inducible nitric oxide synthase-1, interleukin-1, and gro) have NFkappaB binding sites necessary for LPS-induced transcription(9, 10, 11, 12, 13, 14, 15) . When minimal ox-LDL is administered in vivo, MCP-1 is induced in liver cells and NFkappaB is activated(32) , but it is yet to be established whether these effects are observed in hepatic macrophages or in other hepatic cells.

ox-LDL stimulates diverse physiologic and gene effects in macrophages, such as release of prostaglandin E(2) and leukotriene C(4), chemotaxis, endothelial cells adherence, and expression of class II major histocompatibility complex and stress-related genes (5) . ox-LDL also inhibits a number of effects, including expression of several inflammatory genes(6, 7, 8) . The molecular mechanisms by which ligation of SR generates such effects in mononuclear phagocytes remain, however, undefined(3) . The present data indicate that ox-LDL stimulates hydrolysis of PIP(2) and rapid increases in the intracellular concentration of calcium, both of which are inhibited by pertussis toxin. The dual effects of ox-LDL on binding of macrophage extracts to an NFkappaB sequence in an EMSA assay (e.g. induction of a novel band and suppression of a LPS-induced band) are also both pertussis toxin-sensitive. Last, the suppressive effects of ox-LDL on expression of the TNF-alpha gene are also sensitive to pertussis toxin, indicating the gene suppressive effects of ox-LDL are mediated through this signaling pathway.

SR are a group of receptors, expressed principally in macrophages and characterized by high affinity binding of a broad spectrum of molecules, including variously altered proteins(3) . They share this broad, high affinity binding with the LDL receptor-related protein, which is also coupled to a pertussin toxin-sensitive G protein(30) . Well established as major endocytic receptors, SR also modulate a variety of macrophage activities, but intracellular signaling in response to ligation of SR has not been established(3) . The present data indicate that a receptor for ox-LDL is coupled to a pertussis toxin-sensitive G protein, which stimulates hydrolysis of PIP(2), subsequent generation of Ca fluxes, and activation of one or more subunits of NFkappaB. Activation of PIP(2) hydrolysis has been reported to initiate activation and binding of NFkappaB, though the precise molecular mechanism(s) is not established(31) . Induction of NFkappaB complexes by ox-LDL could thus compete with binding of p50-p65 complexes to NFkappaB sites in promoters and thereby inhibit initiation of transcription. A recent report indicates that a cDNA, cloned from the central nervous system of a mollusk, encodes a putative receptor whose extracellular domain has a high similarity to the extracellular binding domain of the low density lipoprotein receptor(33) . The second repeat and C-terminal portion of the putative receptor are quite similar to regions of a specific class of guanine nucleotide binding protein-coupled receptors(33) . Of note, the LDL receptor family, like the scavenger receptors, has a conserved cysteine domain in the extracellular portion(3) .

These observations are potentially of biological interest. Although the interaction of mononuclear phagocytes and ox-LDL is believed to be important in the genesis of atherosclerosis, the exact molecular mechanisms involved remain to be established(1, 5) . In fact, whether macrophages play essentially an active or passive role in this process remains to be determined(7, 34) . If subsequent studies indicate that macrophages do play a passive role (that is are suppressed by ox-LDL from carrying out their proinflammatory duties in atherogenesis), the observations here provide a potential molecular mechanism for this result.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants ES 02922, CA 29589, and HL 24066. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pathology, P. O. Box 3712, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-5709; Fax: 919-684-5215.

(^1)
The abbreviations used are: ox-LDL, oxidized low density lipoprotein; TNF-alpha, tumor necrosis factor-alpha; LPS, lipopolysaccharide; SR, scavenger receptors; PIP(2), phosphatidylinositol 4,5-bisphosphate 2; IP(3), inositol 1,4,5-trisphosphate; EMSA, electrophoretic mobility shift assays.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.