(Received for publication, November 27, 1995)
From the
In order to identify novel genes expressed in macrophage-derived foam cells, we used a multigene assay to examine the expression of genes in control versus cholesterol-loaded macrophages. We compared THP-1 macrophages incubated with or without acetylated LDL (acLDL) ± acyl-CoA:cholesterol O-acyltransferase (ACAT) inhibitor (compound 58035) for 20 h and assessed changes in mRNA of chemokines, growth factors, interleukins, and adhesion molecules. Among 49 genes examined, an increase in mRNA was observed only for interleukin 8 (IL-8) in THP-1 macrophages. Northern analysis confirmed a 3- to 4-fold increase of IL-8 mRNA and an enzyme-linked immunosorbent assay (ELISA) revealed a corresponding increase in IL-8 in conditioned medium. Oxidized LDL (oxLDL) also induced IL-8 mRNA, but native LDL had no effect. 58035 had a moderate effect on IL-8 induction by acLDL. AcLDL-induced IL-8 expression was concentration- and time-dependent. The time course of IL-8 induction paralleled that of cholesterol loading. MCP-1, a chemokine implicated in recruiting monocytes in atherogenesis, was also induced by acLDL. The induction of MCP-1, however, peaked at 1 h after addition of acLDL and returned to basal level by 20 h while IL-8 induction peaked at 8 h and was still 2-fold higher than basal level at 20 h. IL-8 induction was also observed in fresh human monocyte-derived macrophage cells treated with acLDL. Finally, immunohistochemistry and in situ hybridization studies using specimens of human coronary atheromas showed expression of IL-8 mRNA in a macrophage-rich area. We conclude that IL-8 is induced in macrophage foam cells as a response to cholesterol loading. The chemoattractant and/or mitogenic effects of IL-8 on neutrophils, T cells, smooth muscle, or vascular endothelial cells may contribute to the progression and complications of atherosclerosis.
Cholesterol-loaded macrophages are one of the hallmarks of
atherosclerosis(1, 2, 3) . Recent evidence
indicates that foam cells play a major role in the initiation and
clinical complications of atherosclerotic
lesions(4, 5) . Macrophages respond to cholesterol
loading by altering the cellular metabolism and uptake of cholesterol (6, 7) , increasing synthesis of
phospholipids(8) , increasing synthesis and secretion of
apolipoprotein E(9) , and enhancing lipoprotein (a) and
apoprotein (a) internalization and degradation(10) . However,
the altered regulation of other genes not involved in the regulation of
cellular lipid content has also been observed. Macrophage foam cells
show increased expression of tissue factor(11) , matrix
metalloproteases(12, 13) , monocyte chemoattractant
protein 1 (MCP-1, ()14), 15-lipoxygenase(15) , and
other molecules(16) . Although the physiological relevance of
these alterations to foam cell formation is not completely understood,
they probably contribute to different phases in the progression of
atherosclerosis(11, 12, 13, 14, 15) .
Atherosclerosis is a multifactorial, complex pathological process.
Cell-cell and cell-matrix interactions and communications involving
macrophages, vascular smooth muscle cells, vascular endothelial cells,
and lymphocytes are likely to be involved. The identification and
characterization of novel genes involved in this cross-talk could be
crucial to deciphering the mechanisms of atherogenesis. As an approach
to identifying new genes involved in atherogenesis, we have assayed
multiple genes for altered levels of their mRNAs in responses to
cholesterol loading of foam cells. We have identified human IL-8 as an
inducible factor produced by cholesterol-loaded macrophages and have
found that the regulation of its production is correlated with
cholesterol loading. IL-8 is a potent chemokine, but has received
little attention as a potential contributor to atherogenesis.
Human monocyte-derived macrophages (18) were
maintained for 13 days in RPMI 1540, 10% human serum; 1 ng/ml
granulocyte/macrophage-colony stimulating factor was added on days 1,
4, and 11 to up-regulate the scavenger receptor and thus facilitate
cholesterol loading(19) . On day 14, the cells were incubated
with RPMI 1640, 1 Hu-Nutridoma, and 1 ng/ml
granulocyte/macrophage-colony stimulating factor. On day 15, the cells
were incubated with fresh medium; acLDL and/or 58035 was added, and the
experiments were conducted.
The initial multigene assay was performed as
described previously(24) . Briefly, the total RNA isolated from
the cultured cells was reverse-transcribed to generate first strand
cDNA. The second strand of cDNA was synthesized in the presence of
[-
P]dCTP. This pool of labeled cDNA
fragments was hybridized to a nylon sheet on which a known amount of
plasmid DNA from genes of interest had been individually dotted. The
relative mRNA level of each gene was normalized against the signal of
-actin.
Figure 1:
Multigene
assay of gene expression in control versus cholesterol-loaded
macrophages. THP-1 cells were cultured in a serum-free RPMI 1640 medium
with 1 Nutridoma-HU plus 10
M PMA
for 24 h. The medium was replaced by fresh medium with LDL (50
µg/ml), acLDL (50 µg/ml), and compound 58035 (5 µg/ml)
added. After 20 h of incubation, total RNA was isolated from each group
for the assay. A, control; B, LDL alone; C,
acLDL alone; D, acLDL plus 58035. Genes tested were as
follows. Row 1, pUC, TGF
, TGF
, TNF, TIMP2, PDGF-R,
MAX; Row 2, MCP-1, PKC, GAPDH, ECGF
, c-myc,
IGF-1, Ki-ras; Row 3, IL-1R
, IL-1B, IL-2R, IL-3,
IL-4, IL-5, IL-8; Row 4, IGF-2, Rb, p53, IGF-R, CSF-1, FMS,
-IFN; Row 5, MIP-1
, HLA-B, E2F, DR-
, GM-CSF,
DQ-
, EGF-R; Row 6, FN, c-sis, DQ-
, MAC-1,
TIMP-1 ICAM-1, collagenase; Row 7, blank, stromelysin, blank,
gelatinase, blank, VIM, blank: Row 8,
-actin, blank,
IL-6, blank,
-procollagen, blank, cathepsin
D.
Among
49 genes examined, an increase in mRNA was observed only for IL-8 in
acLDL-treated THP-1 cells (Fig. 1). Scanning the filter revealed
no significant alteration in mRNA levels of any other genes tested.
After normalization to -actin, the increase in IL-8 mRNA was
estimated to be 2.1-fold for acLDL-treated cells versus control macrophages. 58035 alone had no effect (data not shown)
but increased IL-8 mRNA by
5-fold when used with acLDL. Native LDL
had a marginal effect on IL-8 mRNA levels, causing increases of 1.2- or
1.4-fold, with or without 58035, respectively. Since phorbol esters
up-regulate scavenger receptors while down-regulating LDL
receptors(27, 28) , these results suggest that IL-8
mRNA is induced as a result of uptake of acLDL by the scavenger
receptors. The synergism between ACAT inhibitor and acLDL could
indicate that accumulation of unesterified cholesterol provides the
signal leading to IL-8 induction.
To confirm these initial results,
Northern blot analysis was performed. Since oxLDL has been shown to
stimulate the production of IL-8 by THP-1 cells without PMA
treatment(30) , we also used oxLDL in the experiment.
Consistent with the results of the multigene assay, the mRNA level of
IL-8 increased 2.5-fold in cells incubated with acLDL alone and
3.5-fold in cells incubated with acLDL plus 58035 compared with
the control cells, as determined by
particle scanning of the
Northern blot shown in Fig. 2. OxLDL also increased IL-8 mRNA
content to a level similar to that of acLDL (Fig. 2). An ELISA
assay was used to determine IL-8 protein levels in cell culture media
collected after lipoprotein treatment (Fig. 3). The accumulation
of IL-8 in medium generally paralleled the changes in mRNA (Fig. 2). In cells incubated with acLDL plus 58035, the medium
IL-8 concentration was
2.4-fold higher than controls.
Figure 2:
IL-8 mRNA
levels in control and cholesterol-loaded THP-1 macrophages.
PMA-pretreated THP-1 cells were incubated for 20 h in medium alone (lane 1) or medium containing 5 µg/ml 58035 (lane
2), 70 µg/ml LDL (lane 3), 70 µg/ml LDL plus
58035 (lane 4), 70 µg/ml acLDL (lane 5), 70
µg/ml acLDL plus 58035 (lane 6), 70 µg/ml oxLDL (lane 7), or 70 µg/ml oxLDL plus 58035 (lane 8).
Total RNA from these cells was extracted, and Northern blot analysis
was performed using cDNA probes to IL-8 and -actin. The band
hybridizing with the IL-8 probe was
1.8 kilobases based upon
relative migration compared with nucleic acid
standards.
Figure 3: IL-8 protein levels in control and cholesterol-loaded THP-1 macrophages. Cells were treated with a procedure similar to that of Fig. 2. Human IL-8 in the culture medium was determined using an ELISA assay. Triple determinations were performed for each group, and the error bar represents standard deviation of the mean.
Bacterial
endotoxin has been demonstrated to be a potent stimulator of IL-8
expression in macrophages(31, 32) . Therefore, the
possibility that these results reflected bacterial endotoxin
contamination of the lipoprotein preparations needed to be addressed.
Several approaches were used to exclude this possibility. First, the
bacterial endotoxin content of the lipoprotein preparation was
determined using the Limulus amoebocyte lysate assay. The
endotoxin content was estimated to be 0.3 pg/µg acLDL protein.
No detectable IL-8 mRNA induction was observed by Northern blot
analysis when THP-1 cells were treated with up to 100 pg/ml LPS alone
(data not shown). Second, polymyxin B, an inhibitor of LPS action on
many types of cells, blocked the effect of LPS (10 µg/ml) on IL-8
induction in THP-1 cells, but had no effect on IL-8 induction by acLDL
(data not shown). Finally, as shown above, native LDL had no effect on
IL-8 mRNA levels, even though the same batch of LDL was used to prepare
the acLDL which did induce the response.
Figure 4:
IL-8 production by macrophages as a
function of acLDL concentration. A procedure similar to that in Fig. 2was used. Triple determinations were performed. Error
bar is standard deviation of the mean, and the asterisk denotes statistic significance at 95% confidence interval based on
Student's t test -
,
-58035;
- - -
,
+58035.
For the time course experiment, we compared the response of
IL-8 and MCP-1. MCP-1, a member of chemokine, has been
demonstrated to be an inducible gene in vascular endothelial cells,
smooth muscle cells, and monocytes upon various stimuli (33, 34) and is expressed in human
atheroma(13, 35) . The acLDL time course experiments
were performed in the presence of 75 µg/ml acLDL and 5 µg/ml
58035. Northern blot analysis was used to follow the alteration of IL-8
and MCP-1 mRNA content of the cells. The results are shown in Fig. 5A, and the mRNA content of IL-8 and MCP-1
(normalized against
-actin mRNA) is quantitated in Fig. 5B. There were marked differences in the kinetics
of the response of IL-8 and MCP-1 mRNA to cholesterol loading. IL-8
mRNA increased gradually and peaked after 8 h of incubation at
6-fold basal level. Then the IL-8 mRNA decreased and at 20 h of
incubation the level was
2.5-fold higher than the basal level. In
contrast, the increase in MCP-1 mRNA could be observed as early as 10
min after incubation, and the peak effect occurred after a 1-h
incubation at 3.5-fold basal level. Subsequently, MCP-1 mRNA content
gradually decreased and returned to basal level 20 h after incubation.
The time course for MCP-1 mRNA explains why the increase of MCP-1 mRNA
was not observed in the multigene assay, since the RNA samples used in
that assay were collected from cells after 20 h of incubation.
Figure 5:
Time course of IL-8 and MCP-1 mRNA
induction and cholesterol loading of macrophages. A, THP-1
cells were incubated with 70 µg/ml acLDL plus 5 µg/ml 58035. At
each time point indicated, total RNA was isolated and Northern blot
analysis was performed using cDNA probes to IL-8, MCP-1, and
-actin. The band hybridizing with the MCP-1 probe was
0.7
kilobase based upon relative migration compared with nucleic acid
standards. B, quantitation of each band of the hybridization
filter as shown in A was performed using a
particle
scanner. IL-8 and MCP-1 signals normalized against that of
-actin
were plotted as a function of the incubation time. C, THP-1
cells were incubated with 70 µg/ml acLDL plus 5 µg/ml 58035. At
each time point indicated, cells were harvested, and cellular free
cholesterol (FC) and cholesteryl ester (CE) were
determined.
To examine the relationship between altered IL-8 mRNA content and accumulation of cellular cholesterol, cellular free cholesterol and cholesteryl ester were determined. As shown in Fig. 5C, the cellular free cholesterol content increased rapidly during the first 8 h of incubation, paralleling the induction of IL-8 mRNA (Fig. 5B). Subsequently, cellular cholesterol content changed little and IL-8 mRNA decreased. In the presence of 58035, the cellular content of cholesteryl ester did not change, reflecting inhibition of ACAT (Fig. 5C).
The time course data suggested that the free cholesterol content of the cell may be related directly to IL-8 mRNA induction in acLDL-treated cells. To further test this hypothesis, THP-1 cells were treated either with free cholesterol or cholesterol incorporated into liposomes in the presence or absence of 58035 in a manner which has been shown to increase the free cholesterol content of macrophages (36) . The IL-8 mRNA level, however, did not show any significant alteration even though THP-1 cellular free cholesterol content was increased up to 3.1-fold over control levels. A possible explanation is that acLDL- and liposome-mediated cholesterol loading of macrophages may result in accumulation of free cholesterol in different cellular compartments and induce different cellular responses. Mechanisms governing cellular cholesterol levels and compartmentation, however, are still largely unknown (see (37) for review).
Next, the effects of a known competitor of scavenger receptor-mediated acLDL binding were assessed. As shown in Fig. 6, IL-8 mRNA induction by acLDL (lane 3) was blocked completely by polyinosinic acid (lane 4), while the competitor alone (lane 2) had no effect, indicating that IL-8 mRNA induction requires scavenger receptor-mediated uptake of acLDL.
Figure 6: Effect of polyinosinic acid on IL-8 induction by acLDL. THP-1 cells were incubated for 20 h with 5 µg/ml 58035 (lane 1), 1 mg/ml polyinosinic acid (lane 2), 70 µg/ml acLDL plus 58035 (lane 3), or 70 µg/ml acLDL plus 58035 and polyinosinic acid (lane 4). Total RNA was isolated and Northern blot analysis was performed.
Figure 7: IL-8 protein and mRNA levels in control and cholesterol-loaded human peripheral monocyte-derived macrophages. A, peripheral monocyte-derived macrophages were incubated for 20 h in medium alone (lane 1) or medium containing 100 µg/ml LDL (lane 2), 100 µg/ml LDL plus 5 µg/ml 58035 (lane 3), 100 µg/ml acLDL (lane 4), 100 µg/ml acLDL plus 58035 (lane 5), or 58035 alone (lane 6). Total RNA was isolated and Northern blot analysis was performed. B, IL-8 in the culture medium was assayed by ELISA. The bar shows the mean value of duplicate determinations.
Thus, IL-8 mRNA is induced by acLDL-mediated cholesterol loading of macrophages in vitro. Nothing, however, is known about the expression of IL-8 mRNA in human atheromatous lesions. To address this question, IL-8 in situ hybridization experiments were carried out using human coronary atherectomy specimens and IL-8 cRNA probes. Monoclonal antibody against smooth muscle actin (SMA) and an antibody against MAC-1, a marker antigen of macrophages, were used to mark cell types in serial sections. Fig. 8shows the results of IL-8 in situ hybridization and cell-specific immunostaining of sequential sections from one specimen. An area with strong hybridization with the antisense cRNA probe against IL-8 mRNA (purple stain, Fig. 8, A and B) also showed heavy staining with a monoclonal antibody against the MAC-1 antigen (brown stain, Fig. 8, C and D), indicating the presence of macrophages and cells expressing IL-8 mRNA in the same area. By contrast, this area showed no signal with the IL-8 sense cRNA probe (data not shown) and did not react with monoclonal antibody against SMA (Fig. 8, E and F) even though an adjacent area was heavily stained by the smooth muscle cell antibody (Fig. 8E). Similar results were obtained on two different atherectomy specimens. These results indicate that IL-8 mRNA is expressed in a macrophage-rich area of the lesion, consistent with expression in macrophage foam cells.
Figure 8:
Sequential human coronary atherectomy
specimen sections localize IL-8 mRNA to individual cells. A,
section labeled by antisense IL-8 digoxigenin-cRNA and visualized by
alkaline phosphatase immunostaining, 20. B,
45
of section in A. C, section labeled by anti-MAC-1 antibody and
visualized by peroxidase immunostaining,
20. D,
45 of section in C. E, section labeled by anti-SMA
antibody and visualized by peroxidase immunostaining,
20. F,
45 of section in E. Counterstained nuclei
can be seen, but no specific staining reflecting anti-SMA antibody
binding is observed. Arrows indicate cells specifically
immunostained by the corresponding method.
This study reveals that cholesterol loading of macrophages by scavenger receptor-mediated endocytosis of acLDL results in increased IL-8 mRNA and protein production. IL-8 appears to be expressed in foam cells in human atheromas. A role of IL-8 in disease processes such as reperfusion injury and adult respiratory distress syndrome has been documented(38) . The present finding suggests the possibility of a role of IL-8 in plaque progression and complications.
Two lines of evidence suggest that IL-8 mRNA induction by acLDL is mediated, at least in part, by an increase in the content of cellular free cholesterol. The effect of acLDL and compound 58035, an ACAT inhibitor, indicates that cellular accumulation of free cholesterol rather than cholesteryl ester is related to IL-8 induction. Further, the time course of cellular free cholesterol accumulation indicates a rapid increase in cellular free cholesterol content which precedes the increase in IL-8 mRNA, also suggesting a close relationship between cellular free cholesterol content and IL-8 mRNA level. Recently, Terkeltaub et al. (30) reported increased production of IL-8 by THP-1 cells when incubated with oxLDL but not with acLDL. The discrepancy is probably due to the fact that nondifferentiated monocytic THP-1 cells were used in that study. Without phorbol ester treatment, these cells have few acLDL receptors(27, 28, 29) . Terkeltaub et al.(30) suggested that oxidative lipid or protein degradation end products or lysophospholipids may contribute to IL-8 induction by oxLDL. We have exercised caution to minimize any oxidation of acLDL preparations used in this study, and acLDL showed a potency similar to that of oxLDL in dose-response experiments. Therefore, oxidative degradation end products cannot account for IL-8 induction by acLDL.
An important finding in this study is the identification of IL-8 mRNA in human coronary atheromatous lesions. The in situ signals indicate a localized expression of IL-8 in the lesions. Due to the nature of the specimen (atherectomy), it is difficult to discern the microtopography of the sections. Nonetheless, immunohistochemical processing of sequential sections with specific monoclonal antibody against macrophage and smooth muscle antigens reveals that the IL-8 mRNA is localized in a distinct macrophage-rich area. It is likely that those cells expressing IL-8 mRNA are macrophages, although double staining would be desirable in future studies for further documentation. Little work has been done beyond the current study to investigate the correlation between IL-8 generation and atherogenesis. In one other study, an increased content of IL-8 protein has been observed in atherosclerotic human abdominal aortic aneurysms compared with normal aortic tissues(39) , although the original source of IL-8 is not addressed in this study.
The multigene assay used in this study to identify IL-8 mRNA induction among multiple genes tested demonstrates the usefulness of this method in detecting altered mRNA levels of genes of interest. As compared with polymerase chain reaction-based differential display, another method commonly used to identify novel genes with altered mRNA levels(40) , this assay has the advantage of simplicity and reproducibility. However, it is evident that changes in unknown genes or low abundance mRNAs would not be detected. Recent studies using expressed sequence tags of PC12 cells before and after treatment with nerve growth factors illustrate that a multitude of cDNAs may be altered when cells are induced to change state(41) . Thus, it is likely that the pattern of altered gene expression in macrophage foam cells is much more complex than documented to date.
Both IL-8
and MCP-1 belong to the chemokine superfamily that has two major
branches based on the structure of the first pair of cysteine residues.
IL-8 has the CXC structure and is a member of the
chemokine family, while MCP-1 maintains a CC sequence and belongs to
the
chemokine family(42) . Due to its monocyte
chemotactic activity, MCP-1 has attracted attention for its potential
role in recruiting monocyte/macrophage cells in atheroma. OxLDL was
recently shown to induce expression by endothelial cells and smooth
muscle cells of MCP-1(33) , and MCP-1 has been identified
within macrophage-derived foam cells in atherosclerotic
lesions(14, 35) . No comparable attention has been
given to IL-8 in atherosclerosis studies, probably because IL-8 was
originally identified as a potent chemotactic factor for neutrophils, a
cell type thought to be relatively uncommon in human atherosclerotic
lesions. However, recent studies demonstrate that IL-8 is a
multifunctional chemokine involved in many biological processes that
could potentially play important roles in atherogenesis. For instance,
IL-8 has been shown to be a potent chemotactic factor for T
lymphocytes(43) . T cells have been identified in human
atherosclerotic lesions although their function is not
clear(44, 45) . Recent studies reveal the presence of
-interferon, a major product of T lymphocytes, in human
atheromatous lesions (46) and suggest its involvement in local
intercellular communications which may be critical in
atherogenesis(47) . IL-8 also has mitogenic and chemotactic
activities toward vascular smooth muscle
cells(48, 49) . A recent study has shown that IL-8
induces proliferation and chemotaxis of human umbilical vein
endothelial cells and is a potent angiogenic agent(50) .
Migration and proliferation of smooth muscle cells are another hallmark
of atherosclerosis, and neovascularization is also a commonly observed
feature of atherosclerotic lesions (see (51, 52, 53) for review). Smooth muscle cell
migration and proliferation is believed to be a major contributor to
atherosclerotic plaque formation, and angiogenesis in atherosclerotic
plaques may predispose to intramural hemorrhage, thrombosis, and plaque
rupture. It is not clear what role, if any, IL-8 plays since many other
protein and peptide factors have been also implicated in regulation of
these processes(51, 52, 53, 54) .
However, findings made through our study and that of Terkeltaub et
al.(30) strongly support a role of IL-8 in atherogenesis.
The potential contribution of IL-8 to the development and complications
of atherosclerosis warrants further investigation.