(Received for publication, December 6, 1994 )
From the
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- (TNF-
). 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
) 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
NF
B sequence oligonucleotide and the LPS-initiated accumulation of
RNA specific for TNF-
. 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
and which can suppress expression of the
TNF-
gene by modulating activation of NF
B.
The interaction of ox-LDL ()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-
, have binding
sites for NF
B in their promoter regions and in fact require these
NF
B sites for transcription (9, 10, 11, 12, 13, 14, 15) .
LPS initiates binding of dimers of the NF
B or Rel homology family
including the NF
B
/Rel A (p50-p65) in extracts of
macrophages and macrophage-like cell lines to oligonucleotides
recognizing NF
B 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 and intracellular Ca
, which are
sensitive to pertussis toxin. Exposure of macrophages to ox-LDL
suppresses LPS-induced activation/binding of NF
B 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 NF
B oligonucleotide and accumulation of mRNA
specific for TNF-
.
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.
When extracts of LPS-stimulated macrophages were examined in
an EMSA against the labeled NFB-binding oligonucleotide, we found
that they induced a distinct retardation band (Fig. 1A, band3 in lane6) comprising
NF
B
/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-
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
NFB. 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 NF
B. 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 NFB(30) , macrophages were exposed to ox-LDL
and examined by digital imaging microscopy for fluxes in
[Ca
]
. Oxidized LDL, in fact,
initiated rapid and transient increases in
[Ca
]
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
, which
was inhibitable by pretreatment of the macrophages with pertussis toxin (Fig. 2B). The increases in
[Ca
]
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).
, macrophage +
native LDL;
, 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
. Native LDL gave no increase in IP
(data not shown). (
, ox-LDL;
, ox-LDL + pertussis
toxin; x ± S.E.).
The effects of
ox-LDL on binding of macrophage extracts to the NFB 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-
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-. 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) .
The data presented here show that ox-LDL inhibits the
LPS-induced binding of extracts of murine tissue macrophages to an
NFB oligonucleotide, which is found in the promoter of the murine
TNF-
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 NF
B
sequences(16, 17, 18, 19) . The
constitutive band generally comprises homodimers of (p50)
and (p52)
. LPS, TNF-
, 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 NF
B oligonucleotide raises the
possibility that the ox-LDL-induced band comprises one or more NF
B
subunits. If so, composition of the novel band could represent a number
of possibilities, including combinations of an NF
B subunit with
similar or additional NF
B subunits, I
B 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-
, inducible nitric oxide synthase-1,
interleukin-1, and gro) have NF
B 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 NF
B 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 and leukotriene C
, 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
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 NF
B
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-
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, subsequent
generation of Ca
fluxes, and activation of one or
more subunits of NF
B. Activation of PIP
hydrolysis has
been reported to initiate activation and binding of NF
B, though
the precise molecular mechanism(s) is not established(31) .
Induction of NF
B complexes by ox-LDL could thus compete with
binding of p50-p65 complexes to NF
B 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.