Regulation of expression of the human monocyte chemotactic protein-1 receptor (hCCR2) by cytokines.

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 γ inhibited MCP-1 binding by 60% at 48 h. The combination of macrophage colony-stimulating factor and interferon γ increased the inhibition to 80% at 48 h. This treatment has been shown previously to induce secretion of tumor necrosis factor α (TNF-α) and interleukin 1 (IL-1) in monocytes. Incubation of THP-1 cells with TNF-α 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-α 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)(3)(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)(13)(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-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% CO 2 atmosphere at 37°C. The cell density was maintained below 0.5 ϫ 10 6 cells/ml.
Equilibrium Binding Analysis-The cells were washed with PBS and suspended at a density of 2 ϫ 10 7 cells/ml in 200 l of binding buffer containing 50 mM Hepes (pH 7.2), 1 mM CaCl 2 , 5 mM MgCl 2 , and 0.5% bovine serum albumin. The cells were incubated with 0.02 nM 125 I-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 125 I-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 Measurement-Cells 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 M 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 ϫ 10 6 cells/ml. After addition of MCP-1 at various concentrations, the elevation of [Ca 2ϩ ] 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 [Ca 2ϩ ] 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 [Ca 2ϩ ] 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 Activity-THP-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 Expression-Total RNA was isolated by the single-step guanidinium thiocyanate-phenol-chloroform extraction (33), and 20 g 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 crosslinking, 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 [␣-32 P]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 ϫ 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 reversetranscribed RNA using Superscript II (Life Technologies, Inc.). Two primers (5Ј-ATGCTGTCCACATCTCGTTCTCG and 5Ј-TTATAAAC-CAGCCGAGACTTCCTG) 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 3Ј-primer showed considerable sequence homology with the 3Ј 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 3Ј-antisense primer and the 5Ј-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 [ 32 P]cDNA hybrids were visualized by autoradiography, and the level of mRNA was quantitated by scanning densitometry.
Statistical Analyses-Data are expressed as mean Ϯ S.D. and analyzed by Student's unpaired two-tailed t test. . This dependence on extracellular Ca 2ϩ 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).

Binding of MCP-1 and Signal
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).
Effect of PMA on MCP-1 Receptor Expression-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 Expression-The requirement of various growth factors for monocyte cell growth, maturation, and differentiation has been well established (38,39). 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 Cytokines-IFN-␥ 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 downregulation 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 Ͻ 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 spindleshaped cells with pseudopodia typical for macrophages became obvious. In contrast, addition of each of the cytokines alone did , and RANTES Indo-1-loaded Mono Mac 6 and THP-1 cells were exposed to the first chemokine (20 nM) and challenged 5 min later by a second dose (20 nM) of the same or different chemokine. Desensitization is indicated by a ϩ and no desensitization is indicated by Ϫ. 3. Time course of PMA-induced reduction of the MCP-1 receptor expression. A, the effect of PMA on specific binding of MCP-1 to monocytic THP-1 and Mono Mac 6 cells was determined. The cells were incubated for up to 6 h at 37°C with PMA at concentrations indicated in parenthesis. The binding of 125 I-MCP-1 was determined in the presence and absence of 100 nM unlabeled ligand, and the specific binding was calculated by subtracting nonspecific binding from the total binding as described under "Experimental Procedures." B, Northern blot analysis was performed on RNA isolated from THP-1 cells exposed to 100 nM PMA for the indicated time periods. 20 g of RNA was analyzed for hCCR2 and ␤-actin transcripts using respective 32 P-labeled cDNA probes as described under "Experimental Procedures." C, the Northern blot was analyzed by scanning densitometry, and the intensity of hCCR2 transcripts were normalized to ␤-actin as a control. not stimulate cell adhesion or any change in cell morphology.

Cell line/first chemokine
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) 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. A, the cells were harvested and washed with PBS and MCP-1 binding analysis was performed using 125 I-MCP-1 (0.02 nM). Specific binding was determined by subtracting nonspecific binding (binding in the presence of 100 nM unlabeled MCP-1) from total binding (binding in the absence of unlabeled MCP-1) as described under "Experimental Procedures." Data are expressed as percent reduction in specific MCP-1 binding compared to untreated control cells. All assays were done in triplicate. *, significantly different from GM-CSF treatment at 6 h and 24 h (p Ͻ 0.05). **, significantly different from MCP-1 treatment at 6 h and 24 h (p Ͻ 0.05). B, total RNA was isolated, reverse-transcribed, and amplified by PCR using primers matching the sequence of the hCCR2 and GAPDH. The amplified DNA was analyzed by Southern blotting using 32 P-labeled cDNA probes specific for the hCCR2 and GAPDH, respectively, as described under "Experimental Procedures."

FIG. 5. Regulation of MCP-1 receptor expression by cytokines.
THP-1 cells were incubated for 6, 24 and 48 h with 1000 units/ml IFN-␥ (IFN), 5 ng/ml TNF-␣ (TNF), 3.2 ng/ml IL-1␣ (IL-1), and combinations thereof (IFN/M-CSF (1000 units/ml, 5 ng/ml) and TNF/IL-1 (5 ng/ml, 3.2 ng/ml)). A, at the indicated time points the cells were harvested and specific MCP-1 binding analysis was performed as described in Fig. 4. Data are expressed as percent reduction in specific MCP-1 binding compared to untreated control cells. All determinations were performed in triplicate. *, significantly different (p Ͻ 0.05). The 6-h time points of all treatments are significantly different from the respective 48-h time points (p Ͻ 0.05) with the exception of TNF treatment. B, total RNA was isolated, reverse-transcribed, and amplified by PCR as described in Fig. 4. The amplified DNA was analyzed by Southern blotting using 32 P-labeled cDNA probes specific for the hCCR2 and GAPDH (GADPH), respectively, as described under "Experimental Procedures."

TABLE II
Effects of cytokine treatment on hCCR2 expression and binding affinity THP-1 cells were treated for 24 h with GM-CSF (5 ng/ml), with IL-1␣ (3.2 ng/ml) and with a combination of TNF-␣ and IL-1␣ (5 ng/ml and 3.2 ng/ml, respectively). Ligand binding analysis was performed by incubating untreated (control) and cytokine-treated cells with various amounts (0.01-40 nM) of 125 I-MCP-1. Maximal binding sites (B max ) and binding affinities were determined by Scatchard analysis using the LIGAND program (30).

fmol/10 6 cells nM
Effect of Neutralizing MCP-1 Antibody on hCCR2 Expression-Leukocytes 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 g/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. DISCUSSION 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 upregulated 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 Ͼ 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 [Ca 2ϩ ] 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.