(Received for publication, August 30, 1996, and in revised form, January 9, 1997)
From the Department of Medicine, University of California, San Diego, La Jolla, California 92093
Monocytes enter the subendothelial space in
response to a variety of chemotactic agents, notably including monocyte
chemotactic protein-1 (MCP-1). To better understand the role of the
human MCP-1 receptor (hCCR2) in monocyte recruitment, we have examined the effects of cytokines on expression of the receptor gene by ligand
binding and Northern blot analysis. THP-1 cells expressed on average
about 5000 MCP-1 receptors/cell. Differentiation of the cells induced
by phorbol myristate acetate resulted in a 75% reduction of receptor
gene expression within 2 h. Macrophage colony-stimulating factor
had only moderate effect on hCCR2 expression. However, interferon inhibited MCP-1 binding by 60% at 48 h. The combination of
macrophage colony-stimulating factor and interferon
increased the
inhibition to 80% at 48 h. This treatment has been shown
previously to induce secretion of tumor necrosis factor
(TNF-
)
and interleukin 1 (IL-1) in monocytes. Incubation of THP-1 cells with
TNF-
and IL-1 induced a rapid down-regulation of hCCR2 expression
and eventual loss of receptor protein. These cytokines exerted their
regulatory role at the level of gene transcription. The effect of
TNF-
alone persisted for 48 h, whereas the cells treated with
IL-1 alone regained all of their receptor activity by 48 h. Our
results suggest that cytokines can profoundly affect the expression of
hCCR2 and thus modulate the recruitment of monocytes into sites of
acute and chronic inflammation, including the developing
atherosclerotic lesion.
Leukocyte recruitment from the circulation into tissues is a complex process and involves a series of molecular events. First, leukocytes adhere loosely to the vascular endothelium. This initial adhesion is reversible unless leukocytes are activated by specific chemoattractants or by cell contact-mediated signals, leading to a tight leukocyte-endothelium adhesion. The subsequent transendothelial migration is then mediated by concentration gradients of chemokines (1). The chemokines are a family of low molecular weight proteins with potent chemoattractant activity for leukocytes sharing significant structural similarities (2-4). Two subfamilies, C-C and C-X-C, can be distinguished based on the absence or presence of an intervening amino acid separating the first two cysteines. Monocytes are selectively attracted by the monocyte chemotactic protein 1 (MCP-1),1 one of the first members of the C-C chemokine family described (5, 6). Human MCP-1 consists of 76 amino acid residues and shows sequence similarity to the mouse competence gene JE (5, 7). In analogy to IL-8, a member of the C-X-C family, the biologically active form is a dimer (8, 9). Analysis of deletion mutants suggests that the amino-terminal region is necessary for binding to the receptor (10, 11).
MCP-1 is produced and secreted by a variety of cells such as vascular
endothelial cells, vascular smooth muscle cells, monocytes, and
fibroblasts in response to specific stimuli. Cytokines appear to play a
crucial role in this cell activation process, and IL-1, IL-4, TNF-,
and IFN-
have been shown to regulate the expression of MCP-1
(12-14). Through a variety of additional stimuli, vascular smooth
muscle and endothelial cells participate in inflammatory and immune
reactions and play an important role in chronic vascular disorders such
as atherosclerosis. Exposure of the vascular endothelium to shear
stress induces the expression of MCP-1 (15) and intercellular adhesion
molecule 1 (16), resulting in recruitment and adhesion of
monocytes. Atherogenic lipoproteins such as oxidized low density lipoproteins accumulate in atherosclerotic lesions (17) and are potent
chemoattractants for circulating monocytes (18). In addition, modified
lipoproteins have been shown to induce the expression of MCP-1 in
cultured human endothelial and smooth muscle cells, thereby augmenting
the inflammatory response (19). In in vivo experiments
it has been shown that MCP-1 is strongly expressed in human and rabbit
atherosclerotic lesions (20), indicating that this chemokine may be
importantly involved in leukocyte recruitment during atherogenesis.
Leukocyte chemotaxis is initiated by the specific interaction of chemoattractants with receptors on the cell surface. High affinity receptors for MCP-1 have been identified on human monocytes (21), as well as on monocytic THP-1 and Mono Mac 6 cells (22, 23). The cDNA for the human receptor has been cloned, and two forms of MCP-1 receptors, receptor A and receptor B, with alternatively spliced carboxyl tails have been identified (23, 24). More recently, the cDNA for the mouse MCP-1 receptor has also been cloned (25, 26). The hCCR2 binds MCP-1 and MCP-3, but does not interact with MCP-2, although all three proteins are structurally related (14, 27). Analysis of the amino acid sequence, the biological response to MCP-1, and the sensitivity to pertussis toxin suggest that the receptor belongs to the family of the seven-transmembrane-spanning, GTP-binding protein-coupled receptors (28, 29).
A large body of information is available on the structure, function, and regulation of expression of MCP-1, but little is known about its receptor. The interaction of MCP-1 with the receptor is essential for monocyte activation and induction of chemotaxis during an inflammatory response. We have, therefore, studied potential mechanisms that may regulate the functional expression of the hCCR2 in monocytes. The effects of growth factors and pro-inflammatory cytokines on hCCR2 gene expression was evaluated to determine the effects on receptor synthesis, and to follow the fate of the receptor during the initial stage of monocyte-macrophage differentiation.
Cell culture medium and Hank's balanced salt
solution were purchased from Life Technologies, Inc. Fetal calf serum
was from Hyclone Laboratories. Recombinant human chemokines, MCP-1,
MIP-1, RANTES, and cytokines, M-CSF, GM-CSF, TNF-
, IL-1
, and
the neutralizing monoclonal anti-human MCP-1 antibody were obtained
from R & D Systems, Inc. IFN-
was obtained from Genentech.
125I-MCP-1 (specific activity, 2200 Ci/mmol) was purchased
from DuPont NEN. Dibutylphthalate and dioctylphthalate were from
Aldrich. PMA was purchased from Sigma.
THP-1 cells were obtained from American Type Culture Collection, and Mono Mac 6 cells were purchased from DST-German Collection of Microorganism and Cell Cultures (Braunschweig, Germany). The cells were grown in RPMI 1640 supplemented with 10% fetal calf serum in a humidified 5% CO2 atmosphere at 37 °C. The cell density was maintained below 0.5 × 106 cells/ml.
Equilibrium Binding AnalysisThe cells were washed with PBS and suspended at a density of 2 × 107 cells/ml in 200 µl of binding buffer containing 50 mM Hepes (pH 7.2), 1 mM CaCl2, 5 mM MgCl2, and 0.5% bovine serum albumin. The cells were incubated with 0.02 nM 125I-MCP-1 and various amounts of unlabeled ligand for 90 min at 25 °C. The incubation was terminated by separating the cells from the incubation buffer by centrifugation through a mixture of dibutylphthalate and dioctylphthalate. The specific binding of 125I-MCP-1 was obtained in direct binding experiments by subtracting from the total binding the nonspecific binding determined in the presence of 100 nM unlabeled MCP-1. All assays were done in triplicate, and the binding data were examined with the LIGAND program (30) or by Scatchard analysis.
Intracellular Calcium MeasurementCells were washed once with PBS containing 1 mg/ml bovine serum albumin, suspended in RPMI 1640 containing 10% serum, and incubated in the dark with 5 µM Indo-1 AM (Molecular Probes) for 30 min at 37 °C. After washing with PBS containing 1 mg/ml bovine serum albumin, the labeled cells were resuspended in Hank's balanced salt solution (containing 1.25 mM calcium) at a density of 0.5 × 106 cells/ml. After addition of MCP-1 at various concentrations, the elevation of [Ca2+]i was monitored by continuous fluorescence measurements using a Perkin Elmer LS50B luminescence spectrophotometer (Perkin Elmer) set to collect data at 400 and 490 nm after excitation at 340 nm essentially as described (31). The concentration of [Ca2+]i was calculated according to Cobbold and Rink (32). For the desensitization experiments, the cells were incubated with 20 nM dose of the first chemokine and challenged after 4 min, when [Ca2+]i reached the basal level again, with a second dose (20 nM) of the same or different chemokine.
Effect of Cytokines on MCP-1 Receptor ActivityTHP-1 cells maintained in RPMI supplemented with 10% fetal bovine serum were washed with PBS and resuspended in RPMI containing 10% fetal bovine serum and a single cytokine or a combination of cytokines, added as indicated. The cytokines were reconstituted in PBS containing 0.5 mg/ml human serum albumin, and the cells were incubated in the presence or absence of cytokines for 6, 24, and 48 h. The effects of the cytokines on hCCR2 expression were determined by ligand binding analysis as described above.
RNA Isolation and Analysis of Gene ExpressionTotal RNA was
isolated by the single-step guanidinium thiocyanate-phenol-chloroform
extraction (33), and 20 µg of RNA was denatured in formamide and
formaldehyde and separated in 1% formaldehyde gels as described (34).
RNA was blotted onto a nylon membrane, immobilized by UV cross-linking,
and the membranes were hybridized and washed prior to autoradiography.
A full-length cDNA of the hCCR2 was amplified by PCR from
reverse-transcribed RNA, which was isolated from untreated THP-1 cells.
The amplified cDNA was sequenced by the dideoxy method (34),
labeled with [-32P]dCTP (3000 Ci/mmol) by the random
priming method (Promega) and used as probe in the hybridization
experiments. The membranes were washed at 68 °C with washing buffer
consisting of 0.1 × SSC and 1% SDS. This stringed washing
condition prevents inexact matching allowing only the formation of
perfect hybrids; therefore, only hCCR2 mRNA was detected. As
control to normalize for differences in the loading and transfer of
RNA, the blot was stripped off and reprobed with labeled cDNA for
actin. In some instances gene expression was also estimated by PCR
amplification of the reverse-transcribed RNA using Superscript II (Life
Technologies, Inc.). Two primers (5
-ATGCTGTCCACATCTCGTTCTCG and
5
-TTATAAACCAGCCGAGACTTCCTG) matching the published sequence of the
human MCP-1 receptor B (23, 24) were used to estimate by PCR the
expression of the receptor gene. The antisense 3
-primer showed
considerable sequence homology with the 3
sequence of other chemokine
receptors such as the human CCR5. However, only hCCR2 was amplified by
PCR using these primers. This was confirmed by including a
hCCR5-specific 3
-antisense primer and the 5
-primer specific for
hCCR2. Under these conditions, no PCR product was detected. In
additional experiments, the PCR products amplified by using the two
hCCR2-specific primers were subcloned and 10 of the subclones were
analyzed by cDNA sequencing. The DNA sequence of all 10 subclones
matched that of hCCR2. As a control GAPDH was amplified under the same
conditions using the appropriate pair of primers (35). The amplified
DNAs were separated in agarose gels, transferred onto nylon membranes,
UV cross-linked, and probed for the hCCR2 and GAPDH using radiolabeled probes prepared as described above. The [32P]cDNA
hybrids were visualized by autoradiography, and the level of mRNA
was quantitated by scanning densitometry.
Data are expressed as mean ± S.D. and analyzed by Student's unpaired two-tailed t test.
THP-1 and Mono Mac 6 cells were incubated with 125I-labeled MCP-1 in the
presence of various amounts of unlabeled MCP-1. As shown in Fig.
1, analysis of the competition binding studies indicated that the specific binding activity of monocytic THP-1 and Mono Mac 6 cell lines for MCP-1 was very similar. In direct binding experiments,
THP-1 cells bound 125I-labeled MCP-1 with a
Kd of 0.53 nM (S.D. ± 0.08 nM) and Mono Mac 6 cells displayed a Kd
of 0.65 nM (S.D. ± 0.10 nM). Analysis of the
binding data using the LIGAND program (30) indicated a single-site
model, which was confirmed by Scatchard analysis (Fig. 1). THP-1 cells
expressed on average 8.4 fmol of receptors/106 cells (S.D. ± 1.3 fmol) compared to 7.9 fmol (S.D. ± 1.3 fmol)/106
Mono Mac 6 cells (Fig. 1). These results, corresponding to about 5000 receptors/cell, are comparable with the binding data established for
human monocytes, which express on an average 1000-3400 receptors/cell with a Kd of 0.9-2.2 nM (10, 21).
The biological activity of the receptor on THP-1 and Mono Mac 6 cells
was further analyzed by induction of transmembrane signaling by MCP-1.
Exposure of the cells to MCP-1 caused a dose-dependent transient increase in intracellular concentration of calcium (Fig. 2). The elevation was immediate, and cells responded to
concentrations of MCP-1 as low as 0.1 nM. The maximal
response was achieved at about 20 nM MCP-1 and was similar
in magnitude in both cell lines. The THP-1 cells appeared to be more
responsive at lower MCP-1 concentrations, showing a half-maximal
calcium response at 0.19 nM (S.D. ± 0.08 nM).
The Mono Mac 6 cells responded to low concentration of MCP-1 with a
more modest elevation of [Ca2+]i displaying an
EC50 of 2.20 nM (S.D. ± 0.20 nM).
When the cells were stimulated with the maximal dose of 20 nM MCP-1, the [Ca2+]i increased on
average from a basal level of 20-25 nM to 305-350
nM. The ligand-induced increase of
[Ca2+]i was dependent on extracellular calcium
and returned to basal level within 4 min after addition of the
chemokine. Stimulation of THP-1 cells with MCP-1 either in calcium-free
medium or in the presence of 10 mM EGTA abolished any
changes in [Ca2+]i (data not shown). This
dependence on extracellular Ca2+ is like that seen in human
monocytes (36), but contrasts with results in transfected human kidney
cells expressing the hCCR2 in which signaling-dependent
mobilization of calcium from intracellular stores occurs (29).
Prior incubation of THP-1 and Mono Mac 6 cells with MCP-1 (20 nM) for 5 min prevented a subsequent response to MCP-1 but
did not cause a heterologous desensitization for either MIP-1 or RANTES (Table I). Similarly, MIP-1
or RANTES did not
cause desensitization to a subsequent stimulus with MCP-1. In contrast,
exposure of human monocytes to MCP-1 has been reported to cause
heterologous desensitization for both MIP-1
and RANTES (22).
|
Differentiation of
monocytes to macrophages induced by PMA leads to a rapid loss in the
chemotactic response to MCP-1 (37). To determine whether
down-regulation of the hCCR2 can account for this loss in response
during the differentiation process, we treated THP-1 and Mono Mac
6 cells with PMA, which decreased the surface expression of the hCCR2
(Fig. 3A). Mono Mac 6 cells appeared more
sensitive to PMA-induced differentiation, and 5 nM PMA
reduced the number of MCP-1 receptors on the cell surface by 80%
within 6 h. Differentiation of THP-1 cells to reduce the number of
surface receptors to comparable levels required 100 nM PMA.
The reduction of binding sites for MCP-1 was accompanied by adherence
of cells to the culture dish.
In order to examine whether the down-regulation of the hCCR2 is a transcriptional or post-transcriptional event, we analyzed RNA isolated from the PMA-treated THP-1 cells by Northern blot analysis (Fig. 3B). Exposure of THP-1 cells to PMA reduced the expression of the hCCR2 gene; within 2 h, the mRNA level decreased by 70%; and after 8 h, essentially no expression was detectable (Fig. 3C).
Effects of Growth and Differentiation Factors on MCP-1 Receptor ExpressionThe requirement of various growth factors for monocyte
cell growth, maturation, and differentiation has been well established (38, 39). The effect of M-CSF and GM-CSF on hCCR2 expression was
investigated by ligand binding and receptor mRNA analysis. Although
THP-1 cells constitutively express the c-fms gene and internalize and degrade M-CSF (40), incubation of THP-1 cells with 5 ng/ml M-CSF for up to 48 h had no effect on the cell surface expression of the receptor. Treatment of THP-1 cells with 5 ng/ml GM-CSF, however, resulted in a significant reduction of the specific binding of MCP-1 (Fig. 4A). To determine the
nature of the reduced expression of hCCR2, we analyzed the cells for
receptor mRNA. As with PMA, GM-CSF reduced the level of hCCR2
mRNA by about 50% in the first 6 h and to barely detectable
levels after 48 h (Fig. 4B). M-CSF had no significant
effect on receptor mRNA levels.
The consequence of prolonged exposure of THP-1 cells to 10 nM MCP-1 itself on the hCCR2 expression was also determined. The effect of MCP-1 was rapid, but appeared to be transient. After an initial decrease by 6 h, the number of MCP-1 receptors on the cell surface was partially restored after 48 h (Fig. 4A). Similar results were obtained when a second dose of MCP-1 (5 nM) was added after 24 h (data not shown). Analysis of the receptor mRNA indicated that the effect of MCP-1 was at the transcriptional level (Fig. 4B).
In contrast, the GM-CSF-induced reduction of the hCCR2 persisted, reflecting the process of differentiation from monocytes to macrophages. GM-CSF induced the formation of clusters of between 5 and 10 cells, but did not stimulate adhesion of the cells to the tissue flask (data not shown). In contrast, cell morphology did not change as a result of M-CSF or MCP-1 treatment. GM-CSF induced a 2.7-fold increase in cell number by 48 h, whereas M-CSF had no effect on cell proliferation.
Regulation of MCP-1 Receptor Expression by CytokinesIFN-
displays potent immunomodulatory effects on a variety of immune cells
and is involved in activation of macrophages. Resting macrophages have
been shown to constitutively express IFN-
, which is up-regulated by
an autocrine mechanism during an immune response (41). We, therefore,
investigated the effect of IFN-
on hCCR2 expression. As shown in
Fig. 5A, exposure of THP-1 cells to IFN-
resulted in a rapid down-regulation of hCCR2 surface expression by
about 40% after 6 h and 60% after 24 and 48 h of treatment.
Present during early macrophage differentiation, IFN-
has been
reported to greatly enhance the secretion of M-CSF (42). Although,
M-CSF by itself had no significant effect on hCCR2 expression in THP-1
cells (Fig. 4), we examined whether it would enhance the effect of
IFN-
. The combination of M-CSF and IFN-
resulted in a reduction
of hCCR2 expression that was slightly but significantly greater than
that in cells incubated with IFN-
alone (80% versus 60%
reduction after 48 h, p < 0.05) (Fig.
5A). This combination also stimulated cell adhesion and colony formation that started after 24 h, and by 48 h more
than 80% of the cells were adherent (data not shown). By 24 h, a
change in cell morphology from the round shape of monocytes to
spindle-shaped cells with pseudopodia typical for macrophages became
obvious. In contrast, addition of each of the cytokines alone did not
stimulate cell adhesion or any change in cell morphology.
IFN- has been reported to augment the M-CSF-induced secretion of
IL-1 and TNF-
in monocytes by 20-fold (43). When we treated THP-1
cells with TNF-
, the binding sites for MCP-1 were reduced by about
80% within 6 h and then remained at the new low level up to
48 h (Fig. 5A). IL-1
also induced a reduction of the
MCP-1 binding sites within the first 6 h, but the inhibitory
effect subsided and the number of cell surface receptors after 48 h was not significantly different from that of the untreated cells
(Fig. 5A). A combination of TNF-
and IL-1
initially
completely abolished the cell surface expression of hCCR2. However, the
degree of inhibition fell slightly but significantly to reach at
48 h the level seen on THP-1 cells treated with TNF-
only.
Presumably this reflects the transient nature of the response to
IL-1
.
In direct ligand binding experiments performed on a separate set of
THP-1 cells treated for 24 h with selected cytokines, we confirmed
that the reduction of MCP-1 binding activity was due to down-regulation
of receptor expression rather than a change in binding activity (Table
II). Untreated control cells displayed a ligand binding
affinity of 0.6 ± 0.1 nM. Treatment of THP-1 cells
with GM-CSF or IL-1 did not change the binding affinity significantly and the cells bound 125I-MCP-1 with an
affinity of 0.9 ± 0.2 and 0.8 ± 0.1 nM,
respectively. The binding sites for MCP-1, however, were reduced by
about 57% in the GM-CSF-treated cells and by about 50% in the cells
treated with IL-1
. Treatment of THP-1 cells with a combination of
TNF-
and IL-1
for 24 h reduced the MCP-1 binding sites by
about 92%, but no significant change in binding affinity was observed.
These experiments indicated, that cytokines reduced the number of cell surface receptors on THP-1 cells without affecting the binding affinity.
|
To determine if the cytokines regulate hCCR2 expression at the level of transcription, we isolated and analyzed RNA from the treated cells. The results shown in Fig. 5B indicate that the decrease of hCCR2 expression was probably a consequence of a reduction of receptor gene transcription.
Effect of Neutralizing MCP-1 Antibody on hCCR2 ExpressionLeukocytes can produce and secrete MCP-1 in response
to stimulation by various cytokines (39). We therefore tested whether cytokines directly or indirectly through secretion of MCP-1 affect hCCR2 expression. THP-1 cells were stimulated for 18 h with
cytokines under established conditions that induced down-regulation of
receptor expression. Neutralizing monoclonal anti-human MCP-1 antibody (10 µg/ml) was added to the cells 10 min prior to the addition of the
cytokines. Cells treated with IL-1, TNF-
, or a combination of
both cytokines displayed an 80-90% reduction in MCP-1 binding (Fig.
6). This down-regulation of hCCR2 expression was not
altered by the presence of anti-human MCP-1 antibody. In contrast, the down-regulation of hCCR2 expression induced by MCP-1 was completely prevented by the antibody. The effects of IFN-
on receptor
expression were only partially (by about 20%) neutralized by the
antibody.
Monocytes play an important role in host defense and are recruited from the blood into tissues in response to inflammatory stimuli such as cytokines and chemokines. Stimulation of the vascular endothelium by cytokines leads to secretion of chemotactic factors such as MCP-1 (12, 13, 44, 45) that can then interact with specific receptors on monocytes mediating transmembrane signaling and inducing chemotaxis.
In the present study, we have demonstrated that certain growth factors and cytokines regulate the level of expression of hCCR2, the MCP-1 receptor, in monocytic cells. THP-1 and Mono Mac 6 cells express functional hCCR2, and although the cell lines may differ in some aspects from human blood monocytes, our data indicated that with the exception of the differences in the heterologous desensitization, the responses of the THP-1 cells used in our experiments were very similar to those reported for human monocytes. In particular, the number of hCCR2, the signal transduction response to MCP-1, and the homologous desensitization of THP-1 cells were similar to that of human monocytes (see "Results"). The major advantage of using THP-1 cells is the homogeneity of the cell line, allowing comparison of findings in different experiments. For this reason, we have used THP-1 cells for the present studies to examine monocytic responses to inflammatory stimuli, even though they are not absolutely identical to human peripheral monocytes.
Differentiation of THP-1 cells induced by PMA resulted in changes of
cell morphology and increased cell adhesion. Concomitantly, the
expression of the hCCR2 gene was down-regulated, suggesting that the
loss of receptor protein is a consequence of monocyte differentiation.
PMA acts by directly activating protein kinase C, bypassing
receptor-mediated signaling, but presumably mimicking responses to a
subset of cytokines. We, therefore, directly tested the effects of
cytokines that lead either to monocyte activation or to
differentiation. M-CSF has been identified as a lineage-specific hematopoetic growth factor that acts directly on monocyte/macrophages and their progenitor cells to stimulate their survival and
differentiation (46). High affinity receptors for M-CSF are encoded by
the c-fms proto-oncogene (47), and its functional expression
has been demonstrated in THP-1 cells (40). However, M-CSF had no effect on the hCCR2 expression in THP-1 cells exposed to the growth factor for
up to 48 h. In addition to M-CSF, IFN- is constitutively expressed in resting macrophages and is up-regulated during an immune
response (41) promoting maturation of human monocytes to macrophages
(42). Low concentrations of IFN-
(10 units/ml) had no effect on
receptor expression; however, higher concentrations (1000 units/ml)
reduced the expression of the hCCR2 significantly. This concentration
of IFN-
has been shown to enhance the basal expression of M-CSF in
monocytes (42). Thus, the increased endogenous M-CSF in combination
with exogenous IFN-
can effectively reduce the hCCR2 gene
expression. To test this hypothesis, we added M-CSF together with
IFN-
to a culture of monocytic THP-1 cells. The receptor expression
was slightly but significantly reduced compared to the effect of
IFN-
alone (p > 0.05). The combination of IFN-
and M-CSF also induced a phenotypic change. THP-1 cells typically grow
in suspension, and addition of M-CSF had no effect; only about 5% of
the IFN-
-treated cells acquired plastic adherence. However, when
IFN-
and M-CSF were added together, 80% of the cells became
adherent by 24 h. Many of the cells appeared spindle-shaped with
pseudopodia and were in contact with other cells.
Stimulation of THP-1 cells with a combination of IFN- and M-CSF
results in a greatly enhanced secretion of TNF-
and IL-1 (43). Both
are inflammatory cytokines that are similar in many of their biological
effects. TNF-
reduced the number of MCP-1 receptors by 80% within
6 h, and the number remained constant thereafter. In contrast,
while IL-1 greatly reduced hCCR2 expression during the first 6 h
of cytokine treatment, the effect was reversible and the cells regained
all of their MCP-1 binding activity within 48 h despite continued
exposure to IL-1. Both cytokines exerted an additive effect on the
receptor expression when added together. Although stimulation of
monocytes can lead to secretion of MCP-1, the effects of TNF-
and
IL-1
on receptor expression occur independently from this event. The
presence of neutralizing anti-human MCP-1 antibody did not change the
effects of these cytokines on MCP-1 binding.
The cellular content of hCCR2 mRNA in response to the above described cytokine treatments essentially followed the same pattern as the receptor protein, suggesting that the cytokine-induced regulation of expression of the hCCR2 is at least in part a transcriptional event. Post-transcriptional events such as altered mRNA stability and turnover, changes in translation, receptor internalization, recycling, and degradation cannot be ruled out.
Short term exposure (5 min) of monocytic cells to MCP-1 led to
desensitization of the receptor preventing subsequent change of
[Ca2+]i. When THP-1 cells were cultured in medium
containing MCP-1 for a prolonged period of time, the number of surface
receptors was significantly reduced. Within 6 h, about 50% of the
receptors were lost from the cell surface. This down-regulation is
transient, and the surface expression of the receptor increases again
after 24 h of MCP-1 treatment. The level of the hCCR2 mRNA was
also reduced in the first 6-24 h and increased thereafter, suggesting that the regulation of receptor density is a transcriptional event and
not due to receptor recycling or translocation to or from an
intracellular pool. Thus by controlling the receptor expression, MCP-1
itself can modulate the inflammatory response over a prolonged period
of time (hours), while desensitization renders the cell non-responsive
to MCP-1 for only a short time (minutes). MCP-1 has been shown to
induce IL-1, but not TNF-, expression in monocytes (48), a finding
that could explain the transient down-regulation of hCCR2, which is
similar in cells treated with IL-1.
In summary, we have shown that THP-1 cells are a suitable model to
study the regulation of the hCCR2 expression. The combination of M-CSF
and IFN induces the synthesis of TNF-
and IL-1, and we have
shown that these cytokines are responsible for the down-regulation of
the hCCR2. The elements that control the receptor gene expression can
be separated from the ones that induce cell differentiation. The
combination of TNF-
and IL-1 completely abolished the receptor gene
expression but did not induce cell adherence or a morphological change
of THP-1 cells. The combination of M-CSF and IFN-
, which did induce
cell differentiation, was much less potent in reducing the receptor
expression, most likely because of the lower concentration of secreted
TNF-
and IL-1. This system, however, contained additional cytokines
or other mediators that induced cell differentiation. In this study, we
have shown that the expression of the hCCR2 gene is modulated by
cytokines such as TNF-
and IL-1. They may play an important
regulatory role during monocyte adhesion and transmigration through the
vascular endothelium under various pathological conditions such as
atherosclerosis.
We thank Simone Green, Nonna Kondratenko, and Jane Wey for excellent technical assistance. We also express our gratitude to Dr. Wulf Palinski, Dr. Daniel Steinberg, and Dr. Joseph L. Witztum for critically reviewing the manuscript.