From the Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406
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ABSTRACT |
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Chemokines play an important role in the
regulation of endothelial cell (EC) function, including proliferation,
migration and differentiation during angiogenesis, and
re-endothelialization after injury. In this study, reverse
transcriptase-polymerase chain reaction was used to reveal expression
of various CXC and CC chemokine receptors in human umbilical vein EC.
Northern analysis showed that CXCR4 was selectively expressed in
vascular EC, but not in smooth muscle cells. Compared with other
chemokines, stromal cell-derived factor-1 (SDF-1
), the known
CXCR4 ligand, was an efficacious chemoattractant for EC, causing the
migration of ~40% input cells with an EC50 of
10-20 nM. Of the chemokines tested, only SDF-1
induced
a rapid, though variable mobilization of intracellular Ca2+
in EC. Experiments with actinomycin D demonstrated that CXCR4 transcripts were short-lived, indicating a rapid mRNA turnover. Interferon-
(IFN-
) caused a pronounced down-regulation of CXCR4 mRNA in a concentration- and time-dependent manner. In
a striking functional correlation, IFN-
treatment also attenuated
the chemotactic response of EC to SDF-1
. IL-1
, tumor necrosis
factor-
, and lipopolysaccharide produced a time
course-dependent biphasic effect on CXCR4 transcription.
Expression of CXCR4 in EC is significant, more so as it and several CC
chemokine receptors have been shown to serve as fusion co-receptors
along with CD4 during human immunodeficiency virus infection. Taken
together, these findings provide evidence of chemokine receptor
expression in EC and offer an explanation for the action of chemokines
like SDF-1
on the vascular endothelium.
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INTRODUCTION |
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The vascular endothelium is strategically located to play a prominent sensory and effector cell role in the maintenance of hemostasis, and during the vascular response to inflammation, infection, and injury (1, 2). The endothelium is also integrally associated with angiogenesis (3) and cardiovascular disorders such as atherosclerosis and restenosis (4). Endothelial cells (EC)1 interact with various inflammatory cells, as well as platelets and smooth muscle cells via a variety of chemotactic factors such as chemokines and their receptors (5, 6).
Chemokines are classified into at least two groups, which differ with
respect to the organization of the dicysteine motif present at the
NH2 terminus. The -chemokines, characterized by the CXC
motif include PF-4, IL-8,
IP-10 and SDF-1. The
-chemokines, characterized by the CC motif include MCP-1, MIP-1
and 1
, and RANTES (5, 7, 8). Chemokines mediate their specific effect on target
cells through two related subfamilies of G-protein coupled receptors.
To date, several CXC and CC functional human chemokine receptors have
been discovered (9-16). In line with their well defined role as
mediators of diapedesis, the chemokine receptors have been
primarily localized on neutrophils, monocytes, lymphocytes, and
eosinophils (5). However, little is known about other distinct functions of these cytokines and their interaction with
non-hematopoietic cells.
Three lines of evidence indicate that human EC also express the genes
for chemokine receptors and thus play an active and important role as
target cells for chemokine function. First, the proliferation,
migration, and differentiation of vascular EC, during angiogenesis, is
modulated by chemokines, apparently via specific receptors. Thus, IL-8
is an inducer of angiogenesis (17), whereas PF-4 (18-20), Gro-
(21), and
IP-10 (22) are inhibitors of EC proliferation and
angiogenesis. Second, it has been suggested that leukocyte adhesion to
the endothelium and transmigration require that chemotactic factors be
immobilized on the EC surface (23, 24). This idea is necessitated due to the obvious conceptual difficulty in generating a chemotactic gradient of soluble chemokines under conditions of blood flow. Although
chemokines can bind cell surface proteoglycans (24, 25), vascular
endothelium may still require expression of receptors that are capable
of immobilizing chemokines to generate a specific haptotactic gradient.
Third, recent studies have shown that CXCR4 (26) and several CC
chemokine receptors like CCR2b, CCR3, and CCR5 (27-30) serve as
co-factors in association with CD4 to permit HIV infection. This also
raises the possibility that the HIV susceptibility of EC in a
CD4-independent manner (31, 32) may be due to their expression of CXCR4
and other chemokine co-receptors. Indeed, evidence for this hypothesis
was provided in a recent study (33), which showed that CXCR4 could
function as an alternative receptor for isolates of HIV-2 in the
absence of CD4.
Therefore, to gain a better understanding of the role of chemokines, we examined the repertoire of chemokine receptor mRNAs expressed by EC. Furthermore, the functional expression and transcriptional regulation of CXCR4 receptor in EC was studied in detail, and its biological implications are discussed.
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EXPERIMENTAL PROCEDURES |
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Materials, Cells, and Culture Conditions--
Recombinant human
IFN-, TNF-
, IL-1
, basic fibroblast growth factor, and
transforming growth factor-
were purchased from Genzyme (Cambridge,
MA). Bacterial LPS, actinomycin D, and Me2SO were from
Sigma. SDF-1
was obtained from Gryphon Sciences (South San
Francisco, CA), and other chemokines were from R&D Systems (Minneapolis, MN).
Oligonucleotides for RT-PCR-- Based on the published sequence of human chemokine receptors, the following pair of consensus degenerate 20-mer primers were synthesized from the ends of the third and seventh transmembrane domains of chemokine receptors.
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RT-PCR with Degenerate Primers and Subcloning of Chemokine
Receptor cDNAs--
Total cellular RNA was isolated from
107 early passage HUVEC and HCAEC by the single extraction
Tri-reagent procedure (Molecular Research Center, Inc. Cincinnati, OH),
according to the manufacturer's protocol and stored dissolved in
Formazol at 80 °C. PCR amplification of total RNA was done with
the GeneAmp RNA PCR kit (Perkin-Elmer) as described previously (34).
Two µg of total RNA was reverse-transcribed with the "downstream"
antisense oligomer, CK-R. The "upstream" oligomer CK-F, was added
directly to the reaction tubes along with the PCR "reaction mix"
and subjected to 35 cycles of amplification. The PCR products were
analyzed on agarose gels and subcloned directly into the
PCRII TA vector (Invitrogen). Plasmid DNA from individual
colonies were analyzed by restriction digestion and sequencing.
Northern Blot Analysis--
Total RNA (10 µg/lane) was
fractionated on 1% agarose-formaldehyde gels, transferred to a nylon
membrane (Amersham Corp.), and covalently linked with a UV cross-linker
(Stratagene Inc., La Jolla, CA). For Northern analysis, 515-base pair
cDNA probes of CXCR1, CXCR2, CXCR3, CXCR4, CCR1, CCR2, and CCR3
were used. The GAPDH gene probe (CLONTECH) was used
to normalize RNA sample differences in each lane. The probes were
labeled with [-32P]dCTP using a random-prime labeling
kit (Promega Corp., Madison, WI) and hybridized overnight at 42 °C
in 6 × SSC buffer (1 × SSC = 150 mM NaCl,
15 mM sodium citrate), 0.1% sodium dodecyl sulfate, 5 × Denhardt's solution, 50% formamide, and 100 µg/ml denatured salmon sperm DNA. Membranes were washed with a final stringency of
0.2 × SSC at 60 °C, and analyzed with a phosphorimager
(Molecular Devices, Inc.) after exposure at room temperature for 3-5
days. Densitometry was used for quantitative analysis.
Flow Cytometric Analysis-- Cell surface expression of CXCR4 receptor was analyzed as described previously (33, 35). Briefly, 5 × 105 HUVEC were permeabilized in the presence of 0.2% Triton X-100/PBS for 2 min, and then resuspended in ice-cold PBS, 0.1% bovine serum albumin. Cells were incubated on ice for 30 min with the primary 12G5 antibody (35) or a control anti-PECAM antibody (R&D Systems) of the same subclass. Cells were then washed twice with ice-cold PBS, 0.1% bovine serum albumin and labeled with a second-stage fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Tago Laboratories). FACS analysis was done with a FACScan flow cytometer (Becton Dickinson).
Ca2+ Mobilization Assay-- For measurements of intracellular calcium [Ca2+]i, EC were loaded with 2 µM fura-2/AM (Molecular Probes, Eugene, OR), rinsed with 1 mM EDTA in Dulbecco's PBS, and resuspended into Krebs-Ringer-Henseleit buffer, pH 7.4, containing 0.1% gelatin. Cells (1 × 106/ml) were stored on ice and diluted for use 1:1 with fresh Krebs-Ringer-Henseleit buffer at 37 °C. Fluorescence of fura-2 in cells was measured with a dual channel fluorometer as described previously (36). Chemokines were added from concentrated stocks in water. To establish the integrity of EC, we also measured [Ca2+]i stimulated by thrombin.
Cell Migration Assay-- HUVEC migration assay was performed using 5 × 105 cells/well (in CS-C medium) in the top chamber of a 6.5-mm diameter, 8-µm pore polycarbonate Transwell culture insert (Costar, Cambridge, MA) as reported previously (37). Incubation was carried out at 37 °C in 5% CO2 for 20 h. After incubation, migrated cells in the lower chamber were counted with a ZM Coulter counter (Coulter Diagnostics, Hialeah, FL). Percent migration was calculated based on the total initial input cells per well.
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RESULTS AND DISCUSSION |
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Chemokine Receptor Expression in EC-- To explore the expression of chemokine receptor transcripts in human EC, total cellular RNA from HUVEC was amplified by RT-PCR with the consensus region primers (see "Experimental Procedures"). An expected 515-base pair cDNA band was amplified and subcloned to generate a cDNA plasmid library enriched for chemokine receptor clones. A total of 110 out of the 250 isolated clones were randomly sequenced and analyzed for their sequence distribution. CXCR4, representing 45% of the sequenced clones was the most prevalent chemokine receptor, followed by clones with identity to CCR3 (10%), the eotaxin receptor. Also present were clones having inserts with CXCR1, CCR1, and CCR2 sequences. These data provide evidence that vascular EC have the ability to express mRNA for several chemokine receptors. The results are also consistent with previous reports where CXCR2 expression was detected in HUVEC by RT-PCR (38), and specific binding of IL-8 and RANTES was observed on the endothelium of postcapillary venules and veins in human skin by using an in situ binding assay (39).
Selective Expression and Regulation of CXCR4 mRNA in Vascular
EC--
Steady state expression of chemokine receptors in vascular EC
was studied by Northern blot analysis of total RNA. Fig.
1A (arrow) shows
that both HUVEC and HCAEC express similar amounts of an expected
1.8-kilobase size mRNA after hybridization with the CXCR4 cDNA
probe. In fact, these results also suggest that CXCR4 is the most
abundant chemokine receptor expressed in vascular EC, as identical
Northern blots with EC RNA did not hybridize with 515-base pair CXCR1,
CXCR2, CXCR3, CCR1, CCR2, and CCR3 cDNA probes (data not shown). It
is conceivable that EC primarily express these chemokine receptors at a
low level, and the binding of chemokines with cell surface
proteoglycans facilitates their interaction with the specific receptors
expressed in low copy numbers. In this context, it is important to note
that several chemokines like IL-8, Gro-,
IP-10, and PF-4 directly
modulate EC proliferation or migration (17-22), presumably in a
receptor-mediated interaction.
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CXCR4 Is Expressed on EC Surface-- The cell surface expression of CXCR4 was evaluated by FACS analysis of HUVEC by using the specific anti-CXCR4 monoclonal antibody 12G5 (35). As demonstrated in Fig. 2, there was a shift in the fluorescence intensity of cells after treatment with 12G5, clearly indicating that mRNA expression of CXCR4 is translated into surface expression of the receptor on HUVEC.
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Determination of Half-life of CXCR4 Transcripts-- To help understand the kinetics of inflammation-mediated transcriptional regulation of CXCR4, we used actinomycin D to determine the half-life of its mRNA. As indicated by the selective degradation of existing mRNA upon addition of actinomycin D to EC cultures (Fig. 3), CXCR4 mRNA has a short half-life of around 2 h and is probably subject to a rapid turnover. This is noteworthy, as such rapid turnover of CXCR4 may allow the EC to respond promptly during conditions of infection and inflammatory stress. In addition we also observed that actinomycin D had the unexpected effect of sharply increasing the steady state levels of CXCR4 mRNA after a short term exposure of only 15-30 min. Many cytokines and cytokine receptors, including CXCR4, have A-U-rich elements in their untranslated regions, which serve as targeting motifs for transcript degradation by specific RNases (41). It is possible that, in addition to its action as a transcriptional inhibitor, actinomycin D also has the unique and immediate effect of imparting stability to existing transcripts of mRNA undergoing rapid turnover.
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Kinetics of CXCR4 Transcription Regulation by Inflammatory
Mediators--
Unlike other inducible chemokines, SDF-1, which is
the known ligand for CXCR4 (42, 43), is constitutively expressed in numerous tissues (8); therefore, its biological action is likely to be
regulated at the level of CXCR4 receptor expression. We examined the
kinetics of cytokine modulation of CXCR4 mRNA expression in EC, and
Northern blots were done to study the effects of IL-1
, IFN-
,
TNF-
, and LPS at different time intervals and concentration ranges.
These mediators are known to be simultaneously up-regulated during
inflammation and the pathogenesis of vascular diseases like
atherosclerosis and restenosis (44), and exhibited distinct effects on
the expression of CXCR4 in EC in the initial studies (Fig. 1). As shown
in Fig. 4, treatment of HUVEC with
IFN-
(103 units/ml) caused a rapid and sustained
decrease in steady state levels of CXCR4 mRNA, which reached its
maximum within 3 h after treatment and continued to exert its
inhibitory effect up to 24 h thereafter. Furthermore, in HUVEC
treated with IFN-
(103 units/ml) for 24 h, there
was a marked reduction in the half-life of CXCR4 mRNA from ~2 h
to about 15 min. Nuclear run-on experiments did not reveal any effect
of IFN-
on the rate of synthesis of CXCR4 mRNA in HUVEC (data
not shown), thereby indicating that its inhibitory effect is caused at
the level of CXCR4 mRNA stability. In contrast to IFN-
,
mediators like TNF-
, IL-1
, and LPS had a distinctly more complex
and unique time-dependent biphasic effect on CXCR4
expression. This effect was characterized by an immediate decrease,
followed by a subsequent reversal and increase in the steady state
levels of CXCR4 mRNA despite continuous exposure of EC to the
cytokines. The mechanism behind this biphasic mode of transcriptional
regulation is unclear at present, although the most likely explanation
is that the extended exposure of EC to TNF-
, IL-1
, and LPS
imparts stability to newly synthesized CXCR4 transcripts that are
otherwise subject to a rapid degradation. In comparison, LPS has been
shown to cause a reduction in mRNA levels of CCR2, CCR1, and CCR5
(45).
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SDF-1 Elicits a Ca2+ Response from EC and Is an
Efficacious and Potent Chemoattractant--
To determine whether EC
express a functional CXCR4 receptor, our subsequent studies used
SDF-1
along with several other chemokines to assess their ability to
induce changes in intracellular levels of Ca2+ and cause
migration. As shown in Fig. 6, SDF-1
induced a rapid elevation of [Ca2+]i in various
EC types, with maximal response at a concentration of 100 nM. In contrast, other chemokines like
-IP10, IL-8,
PF-4, MIP-1
, MCP-1, eotaxin, and RANTES had no effect on EC (data
not shown). Since the Ca2+ flux induced by SDF-1
in
primary cultures of HUVEC and HBMEC was small (50-70 nM)
and characteristically variable, we used FBHEC, an established EC line,
to calculate the EC50 of SDF-1
-mediated response. As
evident in Fig. 6 (inset), SDF-1
induced a robust Ca2+ flux (up to 1 µM) with an
EC50 of ~2 nM in the case of FBHEC.
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IFN- Down-regulates SDF-1
-mediated Chemotactic Response in
EC--
Since CXCR4 expression is sharply down-regulated by IFN-
,
the ability of EC to migrate in response to SDF-1
was studied to
examine the functional consequences of altered gene transcription. Treatment of EC for 24 h with IFN-
(103 units/ml)
produced a significant decrease (>60%) in the number of EC migrating
in response to a SDF-1
gradient (Fig. 7B). This correlates well with the transcriptional down-regulation of CXCR4 observed after treatment with IFN-
.
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ACKNOWLEDGEMENT |
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We thank Dr. J. Hoxie at the University of Pennsylvania (Philadelphia, PA) for the gift of anti-CXCR4 monoclonal antibody 12G5.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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
Cardiovascular Pharmacology, Mail Code UW2511, SmithKline Beecham
Pharmaceuticals, King of Prussia, PA 19406. Tel.: 610-270-5578; Fax:
610-270-5080; E-mail: Shalley_K_Gupta{at}sbphrd.com.
1 The abbreviations used are: EC, endothelial cell(s); CCR, CC chemokine receptor; CXCR, CXC chemokine receptor; FACS, fluorescence-activated cell sorter; FBHEC, fetal bovine heart endothelial cell(s); HUVEC, human umbilical vein endothelial cell(s); HCAEC, human coronary artery endothelial cell(s); HBMEC, human brain microvascular endothelial cell(s); IFN, interferon; LPS, lipopolysaccharide; TNF, tumor necrosis factor; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; IL, interleukin; HIV, human immunodeficiency virus; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RANTES, regulated on activation normal T cell expressed and secreted.
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REFERENCES |
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