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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2004;24:61.)
© 2004 American Heart Association, Inc.

Vascular Biology

C-Reactive Protein Stimulates MMP-1 Expression in U937 Histiocytes Through FcRII and Extracellular Signal-Regulated Kinase Pathway:

An Implication of CRP Involvement in Plaque Destabilization

Takeyla N. Williams; Cecelia X. Zhang; Bryan A. Game; Lin He; Yan Huang

From the Ralph H. Johnson Veterans Affairs Medical Center and Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine Medical University of South Carolina, Charleston, SC.

Correspondence to Yan Huang, MD, PhD, Ralph H. Johnson Veterans Affairs Medical Center and Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, Medical University of South Carolina, 114 Doughty St, Charleston, SC 29403. E-mail huangyan{at}musc.edu

	Abstract

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Objective— It has been shown that plasma level of C-reactive protein (CRP) is an independent predictor for acute coronary syndromes and is associated with plaque weakening. However, the underlying mechanisms are not well understood. In this study, we investigated the effect of CRP on the expression of matrix metalloproteinase-1 (MMP-1) that has been implicated in plaque vulnerability by human U937 histiocytes and monocyte-derived macrophages.

Methods and Results— Enzyme-linked immunosorbent assay of MMP-1 in conditioned medium showed that treatment of U937 cells with 100 µg/mL of CRP for 24 hour led to a 3- to 5-fold increase in MMP-1 secretion. CRP also markedly stimulated MMP-1 release from human monocyte-derived macrophages. In contrast, CRP had no effect on tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion. Northern blot showed that CRP upregulated MMP-1 mRNA expression. Collagenase activity assay showed that CRP increased collagen-degrading activity in cell-conditioned medium. Furthermore, results showed that the stimulation of MMP-1 secretion by CRP was inhibited by anti-CD32, but not by anti-CD64 antibody, and by mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor PD98059. Finally, Western blot showed that CRP stimulated phosphorylation of extracellular signal-regulated kinase.

Conclusions— This study demonstrates that CRP stimulates MMP-1 expression by U937 cells through FcRII and extracellular signal-regulated kinase pathway. These findings suggest that CRP may promote matrix degradation and thus contribute to plaque vulnerability.

Key Words: C-reactive protein • matrix metalloproteinase • arteriosclerosis • gene expression

	Introduction

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C-reactive protein (CRP) is one of acute phase proteins that react to inflammation rapidly.¹ CRP is mainly produced by hepatocytes and its expression is upregulated by inflammatory cytokines such as interleukin-6.¹ Previous studies have well documented that CRP plays a role in host defense against bacterial pathogens and clearance of apoptotic and necrotic cells.² However, recent studies have provided evidence that CRP also has undesirable effects, such as promotion of inflammatory process and inflammation-associated atherosclerosis. For example, several investigations have shown that CRP induces the expression of adhesion molecules and chemokines in human endothelial cells^3–5 and stimulates release of tissue factor from monocytes.⁶ The pathological studies also revealed that CRP is present in atherosclerotic lesions but not in the normal vessel wall.^5,7–9 Furthermore, more than two dozen clinical studies published in the past decade have demonstrated a correlation between plasma CRP levels and acute coronary syndromes.^10–13 All these studies suggest that CRP may be involved in the progression of atherosclerosis.

Plaque rupture and erosion are believed to be the key events that trigger the formation of thrombus and subsequent acute coronary syndromes.^14,15 Because a large number of studies have shown the correlation between plasma CRP levels and acute coronary syndromes,^10–13 the possible role of CRP in plaque vulnerability has been investigated. A recent histopathological study showed that serum CRP level in patients who died of severe coronary artery disease was correlated with the level of immunoreactive CRP in atherosclerotic lesions and the numbers of thin cap atheroma.⁹ Another study using angioscopical examination reported an association between plasma CRP level and the intensity of yellow color in plaques, which is a sign of plaque vulnerability.¹⁶ Although these studies suggest that CRP may play a role in plaque destabilization, the underlying cellular and molecular mechanisms have not been well elucidated.

Recently, we investigated the effect of CRP on matrix metalloproteinase-1 (MMP-1 or interstitial collagenase) expression in human U937 histiocytes. We found that treatment of U937 cells with CRP led to a marked increase in MMP-1 secretion and mRNA expression. Furthermore, we demonstrated that CRP stimulates MMP-1 through Fc gamma receptor II (FcRII) and the extracellular signal-regulated kinase (ERK) pathway. Given the importance of macrophage-derived MMP-1 in weakening of atherosclerotic plaques,^17–19 these findings suggest that CRP may contribute to plaque destabilization.

	Methods

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Cell Culture
U937 histiocytes (American Type Culture Collection) were cultured in a 5% CO₂ atmosphere in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal calf serum. The medium was changed every 2 to 3 days. Histiocyte is also called resident macrophage.²⁰ The histiocytic origin of U937 cells was shown by its capacity for lysozyme production and the strong esterase activity.²¹ Peripheral blood human monocytes were isolated from blood collected from healthy human volunteers as previously described.²² Monocytes were incubated with IMDM containing 30% human serum for 6 days for macrophage differentiation before the treatment with CRP. Human umbilical vein endothelial cells (Cascade Biologics) were cultured in a 5% CO₂ atmosphere in medium 200 (Cascade Biologics) containing 2% fetal bovine serum, 0.17-nmol/L human fibroblast growth factor, 1.6-nmol/L human epidermal growth factor, 2.76-µmol/L hydrocortisone, and antibiotics. The flasks were precoated with 0.1% gelatin. The medium was changed every 2 or 3 days. Cell monolayers in the third and fourth passage were used in the experiments.

ELISA
MMP-1 and tissue inhibitor of metalloproteinase-1 (TIMP-1) secreted into culture medium by U937 cells in response to CRP (Calbiochem, Catalog number: 236608) were quantified using sandwich ELISA kits according to the protocol provided by the manufacturer (R&D System).

Northern Blot Analysis
Total cellular RNA was isolated from U937 cells using the RNeasy minikit according to the instructions from the manufacturer (Qiagen). Northern blot of MMP-1 mRNA was performed as described previously.²³

Collagenase Activity Assay
Collagenase activity in conditioned medium was determined with the EnzChek assay kit according to the protocol provided by the manufacturer (Molecular Probes).

FcR Blocking
To block the binding of CRP to FcRI (CD64) or FcRII (CD32), U937 cells were treated with 100 µg/mL of CRP in the presence of 100 µg/mL or 50 µg/mL of monoclonal anti-CD64 (composition, IgG1,, clone 10.1) or anti-CD32 (composition, IgG2b,, clone FLI8.26, BD PharMingen). Monoclonal antibody clone 27 to 35 (composition, IgG2b,) was used as an isotype control antibody for the anti-CD32 antibody. U937 cell does not possess FcRIII.²⁴

ERK Phosphorylation
Phosphorylation of ERK1/2 was detected by Western blot analysis using monoclonal antiphosphorylated and anti-p42/p44 MAPK antibodies (Cell Signaling Technology) as described.²²

Cell DNA Assay
Cellular DNA was quantified with a CyQUANT cell proliferation assay kit according to the procedures provided by the manufacturer (Molecular Probes).

Statistical Analysis
Data were presented as mean±SEM. Comparison between treatments was performed using ANOVA. A value of P<0.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
CRP Stimulates MMP-1 Secretion and mRNA Expression
Because it has been shown that FcRIIa is the major receptor for CRP in leukocytes²⁵ and our recent studies have shown that both FcRI and FcRII are involved in the upregulation of MMP-1 expression in U937 histiocytes by immune complexes,^22,26 we examined the effect of CRP on MMP-1 secretion by U937 cells. Results showed that 100 µg/mL of CRP stimulated MMP-1 secretion by 3- to 5-fold (Figure 1) and the stimulation was concentration-dependent (Figure I, available online at http://atvb.ahajournals.org). In contrast, CRP had no effect on TIMP-1 secretion (Figure I). Northern blot showed that CRP increased MMP-1 mRNA level (Figure 2), suggesting that the increased MMP-1 secretion from U937 cells in response to CRP is because of the upregulated MMP-1 mRNA expression.

View larger version (15K): [in this window] [in a new window]   Figure 1. Stimulation of MMP-1 secretion by CRP. U937 cells were incubated with 100 µg/mL of CRP for 24 or 48 hours. After the incubation, cell-conditioned medium was collected for ELISA to quantify secreted MMP-1, and cells were lysed for DNA assay as described. Data presented are representative of 3 experiments with similar results.

View larger version (77K): [in this window] [in a new window]   Figure 2. Stimulation of MMP-1 mRNA by CRP. U937 cells were treated with 100 µg/mL of CRP for 24 hours, and total RNA was isolated from cells afterward. Northern blot was performed with 20 µg RNA as described. Data presented are the representative of 2 experiments with similar results.

The effect of CRP on collagenase activity in cell-conditioned medium was also determined by using fluorescein-conjugated type I collagen as a substrate. Results showed that collagenase activity in medium conditioned by CRP-treated cells was significantly higher than that observed with medium conditioned by control cells (Figure II, available online at http://atvb.ahajournals.org). Moreover, our data showed that collagenase activity stimulated by CRP was inhibited by MMP inhibitor 1, 10-phenanthroline (Figure II), indicating that MMPs are responsible for the collagen-degrading activity.

FcRII Is Involved in the CRP-Stimulated MMP-1 Secretion
Because it has been shown that FcRIIa and FcRI are high- and low-affinity receptors, respectively, for CRP in monocytes/macrophages,²⁵ the roles of FcRI and FcRII in the CRP-stimulated MMP-1 expression were determined in our blocking experiments using anti-CD64 (FcRI) and anti-CD32 (FcRII) antibodies. Results showed that 100 and 50 µg/mL of anti-CD32 antibody blocked CRP-stimulated MMP-1 secretion by 76% and 30%, respectively (Figure 3). In contrast, anti-CD64 antibody had no effect. To confirm that anti-CD32 antibody blocks CRP-stimulated MMP-1 secretion by specific binding to FcRII, an isotype control antibody (clone 27 to 35) for the anti-CD32 was used. Results showed that it did not block the stimulation. Thus, these data indicate that stimulation of MMP-1 expression by CRP requires FcRII, but not FcRI.

View larger version (32K): [in this window] [in a new window]   Figure 3. Blocking of CRP-stimulated MMP-1 secretion by anti-CD32 monoclonal antibody. U937 cells were incubated with 100 µg/mL of CRP in the absence or presence of 50 or 100 µg/mL of anti-CD64, anti-CD32, or an isotype control antibody for anti-CD32 for 24 hours. After incubation, the conditioned medium was subjected to ELISA to determine the amount of secreted MMP-1. The amount of secreted MMP-1 by CRP-treated cells in the absence of antibodies (control) was designated as 100%. Data presented are the representative of 3 experiments with similar results.

CRP Stimulates ERK Phosphorylation
In our previous studies, we found that immune complexes, which bind to FcR, stimulate MMP-1 expression through ERK signaling pathway.^22,26 To determine if CRP-stimulated MMP-1 expression is also ERK-dependent, we examined the effect of CRP on the phosphorylation of ERK1/2 in U937 cells by Western blot analysis with the use of both anti-phosphorylated ERK and anti-total ERK antibodies.²² Our data showed that CRP stimulated phosphorylation of ERK, mainly ERK2, in a time-dependent manner with peak phosphorylation occurring at 5 minutes (Figure 4). As control, the cellular level of the total ERK had no change in response to the CRP treatment.

View larger version (31K): [in this window] [in a new window]   Figure 4. Time-dependent stimulation of ERK in U937 cells by CRP. U937 cells were stimulated with 100 µg/mL of CRP for the times indicated and then lysed. Twenty-five micrograms of cell protein were electrophoresed on a 10% SDS polyacrylamide gel and then transferred to a nitrocellulose membrane. The membrane was immunoblotted with anti-phosphorylated or anti-p42/p44 MAPK antibodies as described. ERK was visualized by incubating the membrane with chemiluminescence reagent for 1 minute and exposed to x-ray film for 15 seconds. Data are representative of 3 experiments with similar results.

Stimulation of MMP-1 Secretion by CRP Is ERK-Dependent
We further determined the role of the ERK pathway in CRP-stimulated MMP-1 expression by treating U937 cells with CRP in the absence or presence of PD98059, a specific inhibitor for the mitogen-activated protein kinase/ERK kinase (MEK). Results showed that 50 µmol/L of PD98059 inhibited CRP-stimulated MMP-1 secretion by 90% (Figure 5), suggesting that CRP-stimulated MMP-1 expression is ERK signaling pathway-dependent.

View larger version (16K): [in this window] [in a new window]   Figure 5. Inhibition of CRP-stimulated MMP-1 secretion by PD98059. U937 cells were incubated for 24 hours with 100 µg/mL of CRP in the presence or absence of 50 µmol/L of PD98059. After incubation, cell-conditioned medium was subjected to ELISA to quantify secreted MMP-1. The amount of secreted MMP-1 by CRP-treated cells in the absence of PD98059 (control) was designated as 100%. Data presented are the representative of 3 different experiments with similar results.

Stimulation of MMP-1 Secretion from Human Monocyte-Derived
Macrophages by CRP

To further determine if human macrophages respond to CRP treatment similarly as U937 histiocytes do in MMP-1 secretion, we studied the effect of CRP on MMP-1 release by human monocyte-derived macrophages. Our results showed that CRP treatment markedly increased MMP-1 secretion (Figure 6), indicating that CRP stimulates MMP-1 secretion from both human monocyte-derived macrophages and U937 histiocytes.

View larger version (10K): [in this window] [in a new window]   Figure 6. Stimulation of MMP-1 secretion from human monocyte-derived macrophages by CRP. Monocytes were isolated from human blood and incubated with IMDM containing 30% human serum for 6 days. After the incubation, the cells were treated with or without 100 µg/mL of CRP for 24 hours. Cell-conditioned medium was then collected for ELISA to quantify secreted MMP-1, and the cells were lysed for DNA assay.

CRP Does Not Stimulate MMP-1 Expression in Human Vascular Endothelial Cells
Recent studies have shown that CRP stimulates the expression of adhesion molecule and chemokine in human vascular endothelial cells, indicating that CRP is capable of regulating gene expression through a FcR-independent mechanism. Because human vascular endothelial cells express MMP-1 in a large quantity and the expression is regulated by proinflammatory factors, such as oxidized LDL and cytokines,²³ we determined if CRP stimulates MMP-1 expression in human vascular endothelial cells. Results showed that CRP had no effect on MMP-1 secretion (Figure I). In contrast, phorbol-12 myristate-13-acetate, a positive control, stimulated MMP-1 release.

CRP Treatment Does Not Affect U937 Cell Growth
The effect of CRP on the proliferation of U937 cells was determined by DNA assay. Data showed that treatment of U937 cells with 100 µg/mL of CRP for 24 hours had no effect on cell DNA content (28.4±2.15 for control cells and 30.7±1.07 for CRP-treated cells, P>0.05). In addition, no morphological abnormality was found in cell cultures after CRP treatment. These results suggest that CRP treatment does not affect the growth of U937 cells.

	Discussion

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It is generally accepted that MMPs play an essential role in plaque vulnerability, although the precise mechanisms involved in plaque rupture remain to be defined.²⁷ Among many MMPs implicated in plaque vulnerability, MMP-1 is believed to be important because it is responsible for the initial cleavage of fibrillar collagen type I and III,²⁸ the major matrix components in atherosclerotic plaques.²⁹ The role of MMP-1 in plaque vulnerability is also suggested by its co-localization with macrophages in the shoulder regions where rupture is observed frequently.^17,18 Moreover, animal studies showed that MMP-1 was highly expressed in rabbit atheromatous lesions induced by balloon injury and atherogenic diet, but was downregulated by the treatment with cholesterol-lowering drugs that markedly stabilize the lesions.³⁰ A large number of studies have shown that many atherogenic factors considered to contribute to plaque destabilization, such as shear stress,¹⁸ oxidized LDL,²³ hypercholesterolemia,³⁰ and inflammatory cytokines,³¹ all upregulate MMP-1 expression.

Our present study reports for the first time that CRP stimulates MMP-1 expression in macrophage-like U937 cells and human monocyte-derived macrophages, suggesting that CRP may play a role in plaque destabilization. This notion is also supported by recent pathology studies.^9,32 Torzewski and coworkers reported their detection of CRP in all of the 15 human atherosclerotic lesions they studied and a diffuse CRP staining in the fibromuscular layer of the intima.³² They also demonstrated the positive CRP staining in the majority of macrophage foam cells. Virmani and coworkers showed a diffuse CRP staining in lipid core area and localized CRP staining in the cytoplasm of macrophages.⁹ Furthermore, they also demonstrated a correlation between CRP measured by high-sensitivity assay and the number of thin cap atheroma, suggesting that CRP may promote plaque vulnerability. In this regard, our present study may have elucidated a potential mechanism by which CRP correlates vulnerable plaques.

It has been shown that mild inflammation and viral infections cause elevation of CRP to levels of 10 to 40 µg/mL, although active inflammation and bacterial infection produce levels of 40 to 200 µg/mL.³³ According to our present study, CRP at concentrations from 25 to 200 µg/mL upregulates MMP-1 expression by U937 histiocytes (Figure I). Thus, the elevated CRP concentrations in response to inflammation and infection may be able to provoke macrophages to release more MMP-1. In patients with chronic coronary artery diseases, the plasma CRP concentration only mildly increases and the levels are <10 µg/mL in most cases.¹³ According to our in vitro data, these mild elevations may not be sufficient enough to evoke a significant increase in MMP-1 secretion by macrophages. However, considering that CRP is accumulated in atherosclerotic lesions and may be expressed by cells in the lesions,³⁴ it is possible that the CRP concentration in atherosclerotic plaques could be much higher than that in plasma. McGeer and coworkers reported that the CRP mRNA level in atherosclerotic plaques was 10.2- and 7.2-fold higher than that in normal artery and liver, respectively. By using the reverse transcriptase-polymerase chain reaction and in situ hybridization, they demonstrated that CRP mRNA was expressed by smooth muscle cells and macrophages in the thickened intima of plaques.³⁴ Therefore, it is likely that local CRP levels in atherosclerotic lesions are sufficient to stimulate MMP-1 secretion by macrophages.

We reported previously that both FcRI and FcRII are involved in the stimulation of MMP-1 expression in U937 cells by immune complexes.²² Although these results indicate a coordination between FcRI and FcRII in MMP-1 expression, it is not clear if cross-linking of FcRI or FcRII alone is sufficient to stimulate MMP-1 expression. The present study showed that the engagement of FcRII without FcRI involvement leads to MMP-1 upregulation (Figure 3), indicating that cross-linking FcRII alone is able to stimulate MMP-1. Furthermore, this study seems not to support a previous study reporting that CRP binds to U937 cells via receptors that are distinct from the FcR.³⁵

Our observation that CRP activates ERK is appealing. As it is known that ERK pathway is an important signaling cascade controlling various cellular events, such as cell proliferation, differentiation, migration, and apoptosis,³⁶ this finding suggests that CRP may have direct effects on many biological and pathological processes. Furthermore, this finding also suggests that CRP cross-links Fc receptors because it is known that cross-linking of Fc receptors is required for eliciting signaling pathway.³⁷ Previous studies have shown that although monomeric IgG binds to FcR, it fails to activate FcR-linked signaling pathways because it does not cross-link FcR.³⁷ The finding that CRP activates ERK indicates that CRP behaves differently from IgG in cross-linking of FcR. It is conceivable that the difference may stem from the different molecular structures between CRP, which consists of five subunits and thus five FcR-binding sites,² and monomeric IgG, which has only one FcR-binding site.

In summary, the present study has demonstrated that CRP stimulates MMP-1 expression in U937 cells and the stimulation is FcRII- and ERK-dependent. Because macrophage-derived MMP-1 is believed to destabilize atherosclerotic plaques, this study has revealed a potential role of CRP in the plaque vulnerability.

	Acknowledgments

 
Acknowledgments

This work was supported by a Merit Review Grant from the Research Service of the Department of Veterans Affairs and an Institution Grant from Medical University of South Carolina.

	Footnotes

 
T.N.W. and C.X.Z. contributed equally to this work.

Received September 24, 2003; accepted October 16, 2003.

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