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Originally published online as doi:10.1189/jlb.0505241 on October 21, 2005

Published online before print October 21, 2005
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(Journal of Leukocyte Biology. 2006;79:105-113.)
© 2006 by Society for Leukocyte Biology

Synergistic enhancement of cytokine-induced human monocyte matrix metalloproteinase-1 by C-reactive protein and oxidized LDL through differential regulation of monocyte chemotactic protein-1 and prostaglandin E2

Yahong Zhang and Larry M. Wahl1

Immunopathology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland

1 Correspondence: Immunopathology Section, 30 Convent Drive, Building 30, Room 3A-300, NIDCR/NIH, Bethesda, MD 20892-4352. E-mail: lwahl{at}dir.nidcr.nih.gov


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ABSTRACT
 
C-reactive protein (CRP) and oxidized LDL (ox-LDL) are associated with inflammatory lesions, such as coronary artery disease, in which monocytes and matrix metalloproteinases (MMPs) may play a major role in the rupture of atherosclerotic plaques. Monocytes are recruited to inflammation sites by monocyte chemoattractant protein-1 (MCP-1), which may also participate in the activation of monocytes. The objective of this study was to compare the individual and combined effect of CRP and ox-LDL on human monocyte MMP-1 and the role of MCP-1 in this effect. Although CRP or ox-LDL failed to induce MMP-1 in control monocytes, these molecules enhanced MMP-1 production induced by tumor necrosis factor {alpha} (TNF-{alpha}) and granulocyte macrophage-colony stimulating factor (GM-CSF) with a synergistic increase in MMP-1 occurring in the presence of both mediators. Enhancement of MMP-1 by CRP and ox-LDL was attributable to a differential increase in MCP-1 and prostaglandin E2(PGE2). CRP, at physiological concentrations, induced high levels of MCP-1 and relatively low levels of PGE2, whereas ox-LDL caused a significant enhancement of PGE2 with little affect on MCP-1. Accordingly, CRP- and ox-LDL-induced MMP-1 production by monocytes was inhibited by anti-MCP-1 antibodies and indomethacin, respectively. Moreover, addition of exogenous MCP-1 or PGE2 enhanced MMP-1 production by TNF-{alpha}- and GM-CSF-stimulated monocytes. These results show that the combination of CRP and ox-LDL can cause a synergistic enhancement of the role of monocytes in inflammation, first, by increasing MCP-1, which attracts more monocytes and directly enhances MMP-1 production by activated monocytes, and second, by elevating PGE2 production, which also leads to higher levels of MMP-1.

Key Words: tumor necrosis factor {alpha} • granulocyte macrophage-colony stimulating factor • inflammation • atherosclerosis


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INTRODUCTION
 
Chronic inflammatory lesions are comprised of cells of the immune system and inflammation factors or biomarkers, the composition of which is presumed to determine the pathology associated with these lesions. Monocytes are prominent cellular components in inflammatory diseases, including arthritis, periodontitis, and atherosclerosis, where connective tissue turnover is attributed, in large part, to the matrix metalloproteinase (MMP) family of enzymes. Collectively, the members of the MMP family are capable of degrading all the connective tissue components in the extracellular matrix [1 2 3 ]. A prominent member of the MMP family produced by monocytes/macrophages is MMP-1 (interstitial collagenase), which is involved in the degradation of fibrillar collagens, such as types I, II, and III. We have previously shown that the combination of tumor necrosis factor {alpha} (TNF-{alpha}) and granulocyte macrophage-colony stimulating factor (GM-CSF), cytokines found at inflammatory sites, induces MMP-1 through a prostaglandin (PG)-dependent mechanism [4 ]. MMP-1 has been implicated in the breakdown of the thin fibrous cap over vulnerable atherosclerotic plaques leading to the rupture of these plaques and the subsequent ischemic events. MMP-1 is found in atherosclerotic plaques and shown to colocalize with monocytes/macrophages, prominent cells in the plaque [5 ].

Two major inflammation factors considered as predictors of cardiovascular disease are C-reactive protein (CRP) and low-density lipoprotein (LDL) [6 ]. CRP is an acute-phase protein produced by the liver, which is normally found at low concentrations in the plasma but can increase more than 100-fold in response to inflammatory stimuli [7 8 9 ]. Increased levels of CRP have been associated with atherosclerosis, as well as with other diseases, such as periodontal disease and rheumatoid arthritis [10 , 11 ]. Although CRP is a marker of inflammation, recent evidence indicates that it has a direct role in atherogenesis [12 ]. Moreover, relatively low serum levels of CRP serve as predictors of cardiovascular disease, and <1, 1–3, and >3 mg/L correspond to low, moderate, and high cardiovascular disease risk, respectively [12 13 14 ].

The serum levels of LDL have also been used as a predictor of atherosclerosis. The role of LDL in atherogenesis is believed to be a result of the oxidation of LDL enabling oxidized LDL (ox-LDL) to be taken up by monocyte/macrophages, resulting in the formation of foam cells in the atheroma. In a study comparing these two biomarkers, although CRP was shown to be a stronger predictor of cardiovascular disease than LDL, evaluation of the combination of CRP and LDL proved to be a superior method for risk detection [6 ]. This study indicates that the combination of CRP and LDL may interact in an additive or synergistic manner to induce the pathology associated with atherosclerosis. This conclusion is supported by recent studies in which acute coronary syndrome patients with the lowest levels of CRP and LDL following statin therapy had the least recurrent events or progression of disease [15 , 16 ].

There are several mechanisms through which these biomarkers may initiate and subsequently mediate the pathology associated with atherosclerosis. Initiation of an inflammatory response such as atherosclerosis is believed to begin with the attraction of monocytes to these sites by chemoattractants. Monocyte chemoattractant protein-1 (MCP-1) is a potent and specific chemoattractant for monocytes and is a member of the CC chemokine family and mediates its effects mainly through CC chemokine receptor 2 (CCR2), a G protein-coupled receptor [17 ]. The chemotactic response of monocytes to MCP-1 has been shown to involve several signal transduction pathways [18 19 20 21 ]. The role of MCP-1 in the recruitment of monocytes to atheromas and the development of atherosclerosis has been demonstrated in several transgenic mice overexpressing MCP-1 and in mice deficient in MCP-1 [22 ]. Moreover, high levels of MCP-1 have been found in macrophage-rich atherosclerotic plaques [23 ].

Here, we report that CRP or ox-LDL, when added to cytokine-activated monocytes but not control monocytes, enhanced MMP-1 production through differential mechanisms. CRP mediates its enhancing effects on MMP-1 production, in large part, through a stimulation of significant levels of MCP-1 and relatively low amounts of PGE2, whereas the reverse is true for ox-LDL. This differential regulation of MCP-1 and PGE2 by CRP and ox-LDL, respectively, resulted in a synergistic increase in MMP-1 production when these biomarkers were combined with cytokine-stimulated monocytes.


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MATERIALS AND METHODS
 
Purification of human monocytes
Human peripheral blood cells were obtained by leukapheresis of normal volunteers at the Department of Transfusion Medicine at the National Institutes of Health (NIH; Bethesda, MD). The cells were diluted in endotoxin-free phosphate-buffered saline (PBS) without Ca++ and Mg++ (BioWhittaker, Walkersville, MD) and layered over 20 ml endotoxin-free lymphocyte sedimentation media (ICN Biomedicals, Aurora, OH) in 50 ml tubes (Falcon, Becton Dickinson, Franklin Lakes, NJ). After density separation at 400 g for 30 min, the monocytes in the mononuclear layer were purified by counterflow centrifugal elutriation on a Beckman (Torrence, CA) elutriation system as described previously [24 ], except that pyrogen-free PBS was used in the elutriation procedure. Monocytes were enriched to >90%, as determined by morphology, nonspecific esterase staining, and flow cytometry. Moreover, the purification procedure did not activate the monocytes, as shown by the fact that following overnight incubation at 37°C in suspension, less than 4% of these cells were interleukin-2 (IL-2) receptor-positive, a sensitive marker of monocyte activation [25 ].

Culture conditions and reagents
Purified monocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM; BioWhittaker) supplemented with 2 mM L-glutamine (Mediatec, Washington, DC) and 10 µg/ml gentamicin sulfate (Quality Biological, Inc., Gaithersburg, MD). Reagents used were: CRP (Sigma-Aldrich Co., St. Louis, MO), ox-LDL (Intracel, Frederick, MD), recombinant human MCP-1, TNF-{alpha} (1x107 units/mg), and GM-CSF (1x107 units/mg; PeproTech, Rocky Hill, NJ). CRP was tested for endotoxin contamination by the limulus amebocyte lysate assay, and only those lots were used that contained less than 0.06 ng/highest concentration (40 µg/ml) of CRP used in the experiments. Unless otherwise stated, following purification, the monocytes were adhered for 30 min, and then, CRP was added 30 min prior to the stimulation by TNF-{alpha} and GM-CSF, at which time ox-LDL was also added to some of the cultures. Thirty minutes later, MCP-1 was added to some of the cultures. Each experiment was repeated a minimum of three times with different donors.

Detection of MMP-1 by Western blot analysis
Purified monocytes were cultured at a density of 5 x 106/ml DMEM in 12-well polystyrene plates (Costar, Corning Inc., Corning, NJ). After 36–48 h of stimulation with cytokines, the conditioned media were centrifuged and collected. Bovine serum albumin (BSA; 40 µg/ml) was added to the culture supernatants prior to the precipitation of the proteins with cold ethanol (final concentration, 60%) for at least 15 min at –70°C. The proteins were pelleted by microcentrifuging at 20,800 g for 12 min, washed with ethanol, and lyophilized by rotary evaporation. The lyophilized proteins were resuspended in sodium dodecyl sulfate (SDS)-Laemmli buffer [500 mM Tris-HCl (pH 6.8)/10% SDS/0.01% bromophenol-blue/20% glycerol], reduced with 5% ß-mercaptoethanol, heated for 4 min at 100°C, cooled on ice, loaded, and electrophoresed on a 8–16% Tris-glycine gradient polyacrylamide gel (Invitrogen, Carlsbad, CA) or 10% Tris-glycine gel in SDS running buffer [25 mM Tris-HCl (pH 8.3)/192 mM glycine/10% SDS]. The proteins were transferred onto 0.45 µm nitrocellulose in the buffer containing 25 mM Tris-HCl (pH 8.3)/192 mM glycine/20% methanol and blocked with 50 mM Tris-HCl (pH 7.5)/150 mM NaCl (Tris-buffered saline) containing 5% nonfat dry milk for at least 1 h. For the detection of MMP-1, the blots were incubated overnight with rabbit polyclonal antibodies against MMP-1 [generously provided by Dr. Henning Birkedal-Hansen, National Institute of Dental and Craniofacial Research (NIDCR)/NIH]. Western blots were analyzed by the addition of Alexa Fluor 680 goat anti-rabbit (Molecular Probes Inc., Eugene, OR), and the infrared fluorescence was detected with the Odyssey infrared imaging system (LI-COR, Lincoln, NE). The antibody against MMP-1 recognized the active collagenase (ACL) and procollagenase (PCL) forms. Densitometry analysis of the bands on the Western blots was determined with the LI-COR software analysis program or the ImageQuant software analysis program (Amersham Biosciences, Piscataway, NJ).

Detection of PGE2 and MCP-1 by enzyme-linked immunosorbent assay (ELISA)
PGE2 and MCP-1 levels were determined in the culture supernatants obtained 24 h after stimulation of monocytes with TNF-{alpha} and GM-CSF by ELISA (Neogen Corp., Lexington, KY) according to the manufacturer’s suggestions.

Flow cytometry analysis of CCR2
Purified monocytes were incubated with 10% AB serum for 10 min at room temperature to block Fc receptors for immunoglobulin G (IgG; Fc{gamma}Rs), then washed twice with DMEM, and cultured in suspension at a concentration of 1 x 106/ml DMEM in 5 ml polypropylene tubes. First, anti-human MCP-1 and its anti-mouse IgG2B isotype control (R&D Systems, Inc., Minneapolis, MN) antibodies (10 µg/ml each) were added to the corresponding tubes followed by the addition of CRP (10 µg/ml). TNF-{alpha} and GM-CSF (20 ng/ml each) or ox-LDL (10 µg/ml) were added to some of the culture tubes 20 min later. Following overnight incubation at 37°C, the cells were washed twice with PBS without Ca2+ and Mg2+ (BioWhittaker) containing 0.02% sodium azide (Sigma-Aldrich Co.) and 0.1% BSA (PAB; Irvine Scientific, Santa Ana, CA). For the staining of CCR2, the FcRs were blocked by treatment with 10 µg human IgG/106 cells for 15 min at room temperature (R&D Systems, Inc.) and then incubated with 10 µg phycoerythrin (PE)-conjugated anti-human CCR2 (R&D Systems, Inc.) or 10 µg PE-conjugated mouse IgG2B isotype control (BD Biosciences, San Jose, CA) in a total of 100 µl PAB for 45 min at 4°C. After washing twice with 1 ml PAB, cells were transferred to 5 ml polystyrene tubes, resuspended in 0.5 ml PAB, and analyzed within 24 h or fixed in 0.5 ml PBS containing 2% paraformaldehyde and analyzed 2–4 days later on a FACScan® (Becton Dickinson). Events (104) were collected and analyzed routinely using Lysis II software (Becton Dickinson Immunocytometry Systems, San Jose, CA). The percentage of positive cells and the mean fluorescence intensity of the cells were determined.

Analysis of CCR2 transcripts
Monocytes were cultured in suspension (5x106 cells/ml), and total cellular RNA was isolated at 4, 9, and 15 h after initiation of the cultures with a single-step phenol/chloroform extraction procedure (TRIzol, Invitrogen). Total RNA (2 µg) was reverse-transcribed (RT) by Superscript II (Invitrogen) using 0.12 nmol oligo(dT)10 primer (Roche Molecular Biochemicals, Indianapolis, IN). Expression of the CCR2 gene was determined by semiquantitative polymerase chain reaction (PCR) using a sense primer (5'-ATGCTGTCCACATCTCGTTCTCG) and an antisense primer (5'-TTATAAACCAGCCGAGACTTCCTGC), resulting in a full-length CCR2 {1083 base pairs (bp) [26 ]}. The amplification was carried out for 32 cycles with an annealing temperature of 62°C [27 ]. The RT cDNA in the PCR mixture was used at a concentration that would result in a linear relationship between the template and the product. The amplified DNA was analyzed by agarose gel electrophoresis and stained with SYTO 60 red fluorescent nucleic acid stain (Molecular Probes, Invitrogen Detection Technologies). The intensity of the stained band was analyzed by the Odyssey infrared imaging system (LI-COR) and compared with that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 520 bp), which was used as an internal standard. GAPDH was amplified and analyzed under identical conditions using 5'-TCGGAGTCAACGGATTTGGTCGTA as the sense primer and 5'-ATGGACTGTGGTCATGAGTCCTTC as the antisense primer to normalize relative changes of CCR2 mRNA [26 ]. Additionally, RNA samples, which had not been treated with RT, were analyzed by PCR, and the primers were negative controls for contamination with genomic DNA. These samples were negative for genomic DNA contamination.

Statistical analysis
Comparison between group means was analyzed using ANOVA. The data represent the mean ± SEM. A value of P < 0.05 is regarded as significant.


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RESULTS
 
CRP increases MMP-1 production by cytokine-activated monocytes
The association of CRP and other mediators with inflammation suggests that CRP may act in concert with other inflammatory stimuli to exert its effect on monocyte MMP-1 production. Therefore, we added various concentrations of CRP to monocytes in the presence or absence of TNF-{alpha} and GM-CSF. We have previously shown that TNF-{alpha} and GM-CSF in combination, but not alone, will induce MMP-1 production by monocytes [4 ]. In Figure 1 , we show Western blot analysis of the effect of CRP on MMP-1 production by monocytes obtained from three different donors (A, B, and C). CRP alone failed to induce MMP-1 by monocytes from all donors tested with the exception of Donor C, in which a low level of MMP-1 was detected at 40 µg/ml CRP. However, when CRP was added in the presence of TNF-{alpha} and GM-CSF, it caused a significant enhancement of MMP-1. The CRP-induced increase in MMP-1 was detected at relatively low concentrations, as shown by an increase at 0.2 µg/ml for Donor A and at 1 µg/ml for Donor C, and 10 µg/ml showed a significant increase in MMP-1 in each of the three donors. Varying levels of pro-MMP-1 (PCL; 55 and 53 kDa) versus the active form (ACL; 45 and 43 kDa) were detected in the supernatants of monocyte cultures from different donors. Although there was some donor variability in the CRP dose response, the maximal response ranged from 1 to 10 µg/ml, with a decrease at 40 µg/ml. These findings demonstrate that CRP can cause a significant increase in monocyte MMP-1 production in the presence of TNF-{alpha} and GM-CSF, cytokines found at an inflammatory site.



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  		Figure 1. CRP increases the production of MMP-1 by TNF-{alpha}- and GM-CSF-stimulated monocytes. Purified human peripheral blood monocytes, 5 x 106/ml DMEM, were adhered in 12-well plates for 30 min, and then, CRP was added at the indicated concentrations. TNF-{alpha} and GM-CSF (20 ng/ml each) were added 30 min later to some of the cultures. The 48-h supernatants were assayed for MMP-1 by Western blot analysis. Three experiments are shown with monocytes from different donors (A, B, and C).

Increased MMP-1 production by CRP is mediated, in part, through PGE2
We have previously demonstrated that TNF-{alpha} plus GM-CSF induction of MMP-1 occurs, at least in part, through a PGE2-dependent pathway [4 ]. Moreover, PGE2, in the absence of a primary signal, such as that supplied by cytokines, does not induce MMP-1 production by monocytes. To determine if the increase in MMP-1 caused by CRP in the presence of cytokines was mediated through PGs, indomethacin, a PG inhibitor, was added to monocyte cultures. In Figure 2 , two experiments are shown in which the effects of indomethacin on MMP-1 enhancement by 10 µg/ml CRP and a high concentration of CRP (40 µg/ml) were determined. As shown in both experiments, indomethacin reduced the enhancement of MMP-1 by CRP, which was reversed by the addition of PGE2. These results indicated that CRP modulated PGE2 production by monocytes. Measurement of PGE2 levels in the conditioned media supernatants from three experiments with monocytes from different donors demonstrated that the addition of 10 µg/ml CRP with TNF-{alpha} and GM-CSF versus TNF-{alpha} and GM-CSF caused an increase in the mean level of PGE2 production, which was not statistically significant (Fig. 3 ). However, CRP at 20 and 40 µg/ml, in combination with cytokines, caused a significant increase in PGE2 production. The low levels of PGE2 secreted in the monocyte cultures treated with 10 µg/ml CRP, in contrast to the major inhibition of MMP-1 by indomethcin, may be related to the inability to detect total levels of PGE2 as a result of rapid clearance and/or the action of intracellular levels of PGE2, which may act on nuclear receptors [28 ]. These data demonstrate that the CRP-induced increase in PGE2 by cytokine-stimulated monocytes is partially responsible for the enhanced MMP-1 production.



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  		Figure 2. Inhibition of CRP enhancement of monocyte MMP-1 production by indomethacin and reversal by PGE2. Purified human monocytes, 5 x 106/ml DMEM, were adhered in 12-well plates for 30 min, and then indomethacin (5x10–6 M) was added to some of the cultures 10 min prior to the addition of 10 µg/ml or 40 µg/ml CRP. TNF-{alpha} plus GM-CSF (20 ng/ml each) was added to some of the cultures 30 min later in the presence or absence of the indicated concentration of PGE2. The 48-h culture supernatants were assayed for MMP-1 by Western blot.



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  		Figure 3. CRP enhancement of TNF-{alpha}- and GM-CSF-induced PGE2 production. Purified human monocytes, 3 x 106/0.5 ml DMEM, were adhered in 24-well plates for 30 min, and CRP was added at the indicated µg/ml concentrations. TNF-{alpha} (T) and GM-CSF (G; 50 ng/ml each) were added 30 min later to some of the cultures. The 24-h supernatants were collected and assayed by ELISA for PGE2 production. The PGE2 data obtained from three experiments with monocytes from different donors have been normalized to the controls, which are given a value of onefold. The values are expressed as the mean ± SEM.

Role of CRP induction of MCP-1 in MMP-1 production by cytokine- activated monocytes
The partial inhibition by indomethacin of CRP enhancement of MMP-1 production, as shown in Figure 2 , indicates that CRP may induce additional factors besides PGE2, which account for the increase in MMP-1 by this mediator. Previously, a synthetic peptide of CRP has been reported to stimulate the release of MCP-1 from human monocytes [29 ]. In addition, CRP, at a concentration of 100 µg/ml, has been reported to stimulate the release of MCP-1 by monocytes [30 ]. MCP-1 is a potent chemoattractant that recruits monocytes to sites of inflammation and can also serve as an activator of monocytes. We examined whether the whole CRP molecule at physiological concentrations could be mediating some of its effects on MMP-1 production by monocytes through MCP-1. We initially tested this possibility by adding various concentrations of MCP-1 to monocytes in the presence or absence of TNF-{alpha} and GM-CSF. In Figure 4 , we show Western blot analysis of the effect of MCP-1 on MMP-1 production by monocytes obtained from three different donors (A, B, and C). MCP-1 caused an increase in MMP-1 in the presence of cytokines, but not alone, which was detectable at 0.5 ng/ml, with further enhancement of MMP-1 at higher doses of MCP-1.



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  		Figure 4. MCP-1 enhances the production of MMP-1 by TNF-{alpha}- and GM-CSF-stimulated monocytes (5x106/ml DMEM), which were adhered in 12-well plates for 30 min before the addition of TNF-{alpha} plus GM-CSF (20 ng/ml). MCP-1, alone or at the indicated concentrations, was added 30 min later to the cultures. The 48-h supernatants were assayed for MMP-1 by Western analysis. Results shown are from three experiments with monocytes from different donors (A, B, and C).

Next, MCP-1 neutralizing antibodies were added to monocyte cultures to determine if MCP-1 may function as an additional intermediate in the regulation of MMP-1 production following cytokine plus CRP stimulation of monocytes. MCP-1 neutralizing antibodies, but not control isotype-matched antibodies, partially inhibited TNF-{alpha}- and GM-CSF-stimulated MMP-1 (Fig. 5A ). Moreover, the enhancement by CRP of cytokine-induced MMP-1 was significantly inhibited (from 9.5-fold reduced to 3.5-fold) by neutralizing antibodies against MCP-1. These results indicated that stimulation of monocytes with TNF-{alpha} and GM-CSF induced MCP-1 production, which was further increased by the presence of CRP. Measurement of the MCP-1 levels in the media from three experiments revealed that TNF-{alpha}- and GM-CSF-induced release of MCP-1 was increased by two- to threefold with the addition of 5–20 µg/ml CRP (Fig. 5B) . Thus, although CRP alone had little affect on the levels of MCP-1, when cytokine-activated monocytes are exposed to CRP, there is a significant enhancement in the levels of MCP-1 released into the culture media, which is partially responsible for the increase in MMP-1 caused by CRP.



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  		Figure 5. (A) Antibodies against MCP-1 inhibit cytokine and cytokine plus CRP-induced production of MMP-1. Monocytes were adhered in 12-well plates at 5 x 106/ml DMEM for 30 min, and CRP (µg/ml) was added 30 min prior to TNF-{alpha} (50 ng/ml) and GM-CSF (50 ng/ml). MCP-1 neutralizing antibodies (10 µg/ml) and control isotype-matched antibodies (Control Ab; 10 µg/ml) were added 4 h later to some of the cultures. The 48-h supernatants were assayed for MMP-1 by Western analysis, and the levels of MMP-1 were expressed as fold of intensity as determined by densitometry analysis. (B) Enhancement of cytokine-induced MCP-1 by CRP. Purified human monocytes (3x106/0.5 ml DMEM) were adhered in 24-well plates for 30 min. Then, CRP, at the indicated concentrations (µg/ml), was added 30 min prior to TNF-{alpha} (T) plus GM-CSF (G; 50 ng/ml). The 24-h culture media were assayed by ELISA for MCP-1. The MCP-1 data, obtained from three experiments with monocytes from different donors, have been normalized to the controls, which are given a value of onefold. The data are expressed as the mean ± SEM.

Synergistic enhancement of cytokine-stimulated monocyte MMP-1 by CRP plus ox-LDL is mediated through PGE2 and MCP-1
ox-LDL is another biomarker for vascular disease and potentially other inflammatory diseases. We have previously shown that ox-LDL increases MMP-1 production by cytokine-stimulated monocytes, in part, through a mechanism involving cyclooxygenase-2 (COX-2) and PGE2 but does not induce MMP-1 production when added alone to monocytes [31 ]. As monocytes may be exposed to ox-LDL and CRP at an inflammation site, we compared the individual versus the combined effect of these two biomarkers on MMP-1 production as regulated by PGE2 and MCP-1. Addition of ox-LDL or CRP at 1 µg/ml with cytokines caused a 3.9- and 1.3-fold increase in MMP-1 production above cytokines alone, respectively, and when ox-LDL and CRP were added together at 1 µg/ml, there was a 7.9-fold increase in MMP-1 (Fig. 6 ). At 10 µg/ml, ox-LDL enhanced MMP-1 production by 16-fold and CRP, by fourfold, with a synergistic increase of 31-fold above cytokines alone when they were added to the cultures together at 10 µg/ml each. Thus, in comparison with TNF-{alpha} plus GM-CSF alone, the additional exposure to the combination of ox-LDL and CRP caused a striking increase in MMP-1 production. These findings show that exposure of cytokine-stimulated monocytes to the combination of ox-LDL and CRP results in a synergistic increase in MMP-1 production.



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  		Figure 6. CRP and ox-LDL cause a synergistic increase in TNF-{alpha}- and GM-CSF-induced MMP-1 production. Following adherence of purified human monocytes (5x106/ml DMEM) in 12-well plates for 30 min, CRP and ox-LDL were added alone or in combination at the indicated concentrations, followed by the addition of TNF-{alpha} plus GM-CSF (20 ng/ml) 30 min later. The 48-h culture media were assayed for MMP-1 by Western analysis, and the levels of MMP-1 were expressed as fold of intensity, as determined by densitometry analysis. The data were normalized to TNF-{alpha} and GM-CSF, which were given a value of onefold, and the numbers in the open, stacked bars represent the fold increase above TNF-{alpha} and GM-CSF.

As previously shown in Figure 2 , PGE2 levels influence the production of monocyte MMP-1. Therefore, we examined the effect of CRP plus ox-LDL on PGE2 production to determine if that may account, in part, for the increase in MMP-1 production by the combination of these biomarkers. Figure 7 shows data from ELISA analysis of PGE2 in three monocyte experiments from different donors, which have been normalized to the levels of PGE2 in control cultures given a value of onefold. When cytokine-stimulated monocytes were exposed to ox-LDL or CRP at 10 µg/ml, ox-LDL enhanced PGE2 approximately twofold compared with cytokines alone, whereas CRP caused only a slight increase in the mean value for PGE2, which was not significant. When ox-LDL and CRP were combined, there was a further increase in the mean level of PGE2, which was not significant. These results demonstrate that ox-LDL, at relatively low concentrations, is more effective in the stimulation of PGE2 by cytokine-activated monocytes than CRP.



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  		Figure 7. Effect of CRP and ox-LDL on TNF-{alpha}- and GM-CSF-induced PGE2 production. Purified human monocytes (3x106/0.5 ml DMEM) were adhered in 24-well plates for 30 min, and then CRP (10 µg/ml) and ox-LDL (10 ug/ml) were added alone or together in the presence or absence of TNF-{alpha} (T) plus GM-CSF (G; 50 ng/ml). The 24-h culture media were assayed for PGE2 by ELISA. The data obtained from three experiments with monocytes from different donors have been normalized to the controls, which are given a value of onefold. The values are expressed as the mean ± SEM.

As CRP mediated some of its effects through MCP-1, ox-LDL was also examined for its ability to increase MCP-1 in the presence or absence of CRP. Addition of ox-LDL to cytokine-stimulated monocytes resulted in a slight increase in MCP-1, which was not significant, whereas CRP caused a twofold increase in MCP-1 (Fig. 8 ). However, the combination of ox-LDL and CRP resulted in a synergistic increase of threefold in MCP-1, which was significant (P<0.01) compared with cytokines and CRP. Thus, ox-LDL and CRP act synergistically to enhance MCP-1.



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  		Figure 8. CRP and ox-LDL cause a synergistic increase in monocyte MCP-1 when added with TNF-{alpha} and GM-CSF. Purified human monocytes (3x106/0.5 ml DMEM) were adhered in 24-well plates for 30 min, and then, CRP (5 µg/ml) was added in some of the cultures for 30 min followed by the addition of ox-LDL (5 µg/ml), TNF-{alpha} (T; 50 ng/ml), and GM-CSF (G; 50 ng/ml). The 24-h culture media were assayed for MCP-1 by ELISA. The data are the mean ± SEM of three experiments with monocytes from different donors.

These data indicated that CRP and ox-LDL mediated their enhancing effect on MMP-1 production by cytokine-stimulated monocytes, primarily through MCP-1 and PGE2, respectively, but can act synergistically in the induction of MCP-1 and MMP-1. To examine the extent to which PGE2 and MCP-1 contributed to the enhancement of MMP-1 production by monocytes stimulated with cytokines and CRP plus ox-LDL, the cultures were treated with indomethacin in the absence or presence of neutralizing antibodies against MCP-1. As shown in Figure 9 , indomethacin decreased the production of MMP-1 caused by the combination of cytokines and CRP plus ox-LDL. When antibodies against MCP-1 were added with indomethacin, there was a further inhibition of MMP-1, whereas the control isotype IgG had no effect. These results demonstrate that PGE2 and MCP-1 play distinct roles in the regulation of MMP-1 production by monocytes.



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  		Figure 9. MCP-1 enhancement of monocyte MMP-1 involves a mechanism that is independent of PGs. Purified human monocytes (5x106/ml) were adhered in 12-well plates for 30 min, and then, indomethacin (5x10–5 M) and CRP (10 µg/ml) were added 30 min prior to TNF-{alpha} plus GM-CSF (20 ng/ml each) and ox-LDL (10 µg/ml). Four hours after the addition of cytokines, neutralizing antibodies (5 µg/ml) against MCP-1 and isotype control IgG antibodies (IsoIgG; 5 µg/ml) were added to the cultures. The 48-h conditioned media were assayed for MMP-1 by Western blot, and the levels of MMP-1 were expressed as fold of intensity as determined by densitometry analysis. The data are normalized to TNF-{alpha} and GM-CSF, which are given a value of onefold.

Stimulation of monocytes with cytokines, CRP, and ox-LDL does not increase CCR2 expression or receptor levels
A potential explanation for the enhancement of MMP-1 production by MCP-1 may be an increase in the message and receptor levels of CCR2 following stimulation with cytokines and/or CRP and ox-LDL. However, the mRNA transcripts for CCR2 were actually decreased following stimulation of monocytes with cytokines, and a further reduction was with the addition of CRP and ox-LDL (data not shown). Our findings are in agreement with previous reports in which ox-LDL was shown to down-regulate CCR2 expression in the THP-1 cell line [26 ]. The expression and receptor levels of CCR2 in monocytes were also decreased by CRP, which is in contrast to the observed increase in CCR2 expression following treatment of the THP-1 cell line with CRP [32 ]. This difference may be related to functional responses that differ between primary monocytes and monocytic cell lines. Thus, the increase in MMP-1 mediated by MCP-1 is not a result of an up-regulation of CCR2 by CRP or ox-LDL.


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DISCUSSION
 
Inflammatory lesions are associated with multiple biomarkers, which may act in concert to initiate the pathology associated with these diseases. CRP and LDL have been shown to be biomarkers for the risk of cardiovascular disease [6 ]. A study of ~28,000 women has shown that CRP is a stronger predictor of cardiovascular events than LDL. However, the highest risk of cardiovascular events occurs when both factors are high. Further evidence for this conclusion comes from recent studies in which acute coronary syndrome patients who have the lowest levels of LDL and CRP following statin treatment have the greatest reduction in progression of the disease and recurrent events [15 , 16 ]. Moreover, these studies indicate that LDL and CRP function as distinct entities to bring about the combined effect on recurrent events as demonstrated by better clinical outcomes with lower CRP levels regardless of the LDL cholesterol level.

It is believed that one mechanism for the ischemic events involved in atherosclerosis is a result of the rupture of vulnerable plaques, those that have a thin, connective tissue cap, as a result of the action of MMPs produced by monocytes/macrophages, a predominant cell of the atheroma, and smooth muscle cells. Our findings demonstrate that exposure of TNF-{alpha}- and GM-CSF-activated monocytes to CRP or ox-LDL at physiological concentrations increased the levels of MMP-1. However, a synergistic increase in MMP-1 occurred when CRP and ox-LDL were present. Moreover, the synergistic increase in MCP-1 induced by CRP and ox-LDL acts as an intermediate signal, which is partially responsible for the enhancement of MMP-1 production by monocytes. The role of MCP-1 as an intermediate was demonstrated by partial inhibition of MMP-1 by antibodies against MCP-1 and by an increase in MMP-1 production by the exogenous addition of MCP-1 to activated monocytes.

CRP and ox-LDL have been reported to affect a number of monocyte functions. Monocyte activities enhanced by CRP include phagocytosis [33 , 34 ], tumoricidal activity [35 ], respiratory burst [36 ], production of hydrogen peroxide [37 ], and secretion of the cytokines TNF-{alpha}, IL-1, IL-6, and MCP-1 [29 , 38 , 39 ]. In contrast to our findings that CRP caused a decrease in CCR2 in monocytes, CRP has been reported to increase CCR2 in the THP-1 cell line [32 ]. This difference may be a result of functional differences between primary monocytes and monocytic cell lines. CRP, at a high concentration (100 µg/ml), has also been reported to induce MMP-1 production directly by U937 histiocytes and monocyte-derived macrophages [40 ]. Our findings failed to show direct stimulation of monocyte MMP-1 by CRP at physiological concentrations. However, CRP, at concentrations of 1–10 µg/ml, caused a significant enhancement of MMP-1 production by cytokine-stimulated monocytes. These doses of CRP correlate with the serum levels of CRP, which serve as predicators of cardiovascular disease, and <1, 1–3, and >3 mg/L correspond to low, moderate, and high cardiovascular disease risk, respectively [12 13 14 ]. At a higher dose of 40 µg/ml, a decrease in optimal production of MMP-1 was noted. We are currently investigating the reason for this biphasic effect. One possibility is that higher concentrations of CRP change the ratio of p38 to extracellular signal-regulated kianse (ERK)1/2 activation. Our previous studies and unpublished findings have shown that p38 can function as a negative regulator of ERK1/2 and through this mechanism, decrease MMP-1 production [41 ].

Like CRP, ox-LDL also enhances functional responses of monocytes. ox-LDL has previously been shown to regulate MMP production by monocytes/macrophages. ox-LDL caused an increase in MMP-9 production by monocyte-derived macrophages [42 ], enhanced MMP-1 and MMP-9 in lipopolysaccharide (LPS) or cytokine-stimulated monocytes through a COX-2/PGE2-dependent mechanism [31 ], and increased membrane type 3–MMP production by cytokine-stimulated monocyte-derived macrophages [43 ]. ox-LDL has also been shown to enhance macrophage MCP-1 expression and protein in rabbit macrophages [44 ]. In general, ox-LDL is effective in regulating most monocyte/macrophage functions, only in the presence of costimulants, such as LPS or cytokines [31 , 43 , 45 46 47 ].

CRP and ox-LDL mediate their effects through the interaction with several putative receptors. Studies about CRP binding to monocytes have shown that CRP binds to FcRIIa with high affinity and to Fc{gamma}RI with lower affinity [48 , 49 ]. ox-LDL interacts with scavenger receptors (SRs) on the cell surface, which include class A SR [50 ], class B SR (CD36) [51 ], class B SR type-I [52 ], and lectin-like, ox-LDL receptor-1 [53 ].

A synthetic peptide from CRP has been shown to enhance MCP-1 directly by human monocytes [29 ], and whole CRP at a concentration of 100 µg/ml has been reported to stimulate MCP-1 [30 ]. We have shown that the whole CRP molecule at physiological concentrations enhances MCP-1 by monocytes, only in the presence of activating cytokines. Moreover, CRP induces substantially higher levels of MCP-1, whereas ox-LDL was relatively ineffective in increasing MCP-1. However, when monocytes are exposed to ox-LDL in combination with CRP in the presence of cytokines, there is a synergistic increase in MCP-1 production. In addition to being a potent chemoattractant, MCP-1 also affects monocyte activation, as we show by its intermediate role in the enhancement of MMP-1 production by monocytes. MCP-1 has also been shown to induce SR expression through the ERK pathway [54 ], which would potentially increase the sensitivity of monocytes to ox-LDL. The signaling mechanism by which MCP-1 enhances MMP-1 production is currently being investigated. Thus, the increased levels of MCP-1 production by monocytes in response to CRP and ox-LDL would lead to the accumulation of larger numbers of monocytes at an inflammatory site as well as serve as an intermediate in the enhancement of MMP-1 production.

In summary, our data demonstrate for the first time that the combined interaction of CRP plus ox-LDL with monocytes primed with inflammatory cytokines results in a synergistic enhancement of MMP-1 production, which is mediated by PGE2 and MCP-1. Moreover, this is the first demonstration that MCP-1 can act as an intermediate in the induction of monocyte MMP-1. These findings provide further insight into the potential role of CRP and ox-LDL in the pathology associated with atherosclerosis and other inflammatory lesions.


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ACKNOWLEDGEMENTS
 
This research was supported by the Intramural Research Program of the NIH, NIDCR. We thank Drs. Nancy McCartney-Francis and Wanjun Chen for their review of the manuscript.

Received May 5, 2005; revised August 9, 2005; accepted August 31, 2005.


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