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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2533.)
© 2000 American Heart Association, Inc.

Vascular Biology

Fc- Receptor Cross-Linking by Immune Complexes Induces Matrix Metalloproteinase-1 in U937 Cells via Mitogen-Activated Protein Kinase

Yan Huang; Andrew J. Fleming; Shan Wu; Gabriel Virella; Maria F. Lopes-Virella

From the Division of Endocrinology, Diabetes, and Medical Genetics (Y.H., A.J.F., S.W., M.F.L.-V.), Department of Medicine, and the Department of Immunology and Microbiology (G.V.), Medical University of South Carolina, and the Ralph H. Johnson Veterans Administration Medical Center (Y.H., M.F.L.-V.), Charleston, SC.

Correspondence to Yan Huang, MD, PhD, 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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Abstract—Matrix metalloproteinase-1 (MMP-1) secreted by macrophages potentially contributes to plaque rupture. Because large quantities of immunoglobulin G and ICs (ICs), including low density lipoprotein–containing ICs (LDL-ICs), are present in atherosclerotic lesions, we examined the effect of LDL-ICs on macrophage MMP-1 expression. With the use of ICs prepared with human LDL and rabbit anti-LDL antiserum, our enzyme-linked immunosorbent assays and Northern blots showed that MMP-1 secretion and expression by U937 histiocytes were induced by LDL-ICs. Furthermore, our results showed that blocking of Fc- receptor I and II (FcRI and FcRII) inhibited 70% and 55%, respectively, of the LDL-IC–induced secretion of MMP-1. Finally, our data showed that both PD98059, an inhibitor of the mitogen-activated protein kinase pathway, and Ro-31-2880, an inhibitor of protein kinase C, inhibited LDL-IC–stimulated MMP-1 secretion in a dose-dependent manner, with 96% and 95% inhibition, respectively, at the respective doses of 50 µmol/L and 80 nmol/L. In conclusion, this study demonstrated for the first time that LDL-ICs induce macrophage MMP-1 secretion by cocross-linking FcRI and FcRII and triggering a protein kinase C–dependent mitogen-activated protein kinase pathway. These results suggest that LDL-ICs and other ICs localized in atherosclerotic plaques may be potent stimulators for macrophage MMP-1 expression and may contribute to plaque rupture and acute coronary events.

Key Words: LDL • metalloproteinase • immune complex • collagen

	Introduction

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Matrix metalloproteinases (MMPs) have been implicated in the disruption of atherosclerotic plaques, which leads to acute coronary artery events.^{1 2 3} In the MMP family, MMP-1 is responsible for the initial cleavage of fibrillar type I collagen,^{4 5} which accounts for 50% to 75% of total collagen in the intima of atherosclerotic plaques.^{6 7} Immunocytochemistry studies have shown that MMP-1 is expressed by all 3 major cellular components in atherosclerotic lesions: endothelial cells, smooth muscle cells, and macrophages, but not by cells in the normal arterial wall.⁸ Focal overexpression of MMP-1 in human atherosclerotic lesions was frequently found in areas that would be anticipated to have increased mechanical stress and be prone to rupture.⁴ These data strongly suggested that MMP-1 might play an important role in the vulnerability of atherosclerotic plaques.

Plaque rupture occurs most frequently at the shoulder regions of atherosclerotic plaques and results in hemorrhage, thrombosis, and rapid occlusion of the artery.⁹ On the basis of the observation that immunoreactive MMP-1 and macrophages are colocalized in the ruptured shoulder regions, it is generally believed that macrophages are the major source of MMP-1 contributing to plaque rupture.^{4 10 11 12 13} Inflammatory cytokines have been postulated to be responsible for inducing MMP-1 expression in the lesion-associated macrophages, because cytokines such as tumor necrosis factor- TNF and interleukin-1ß IL-1ß have been detected in atherosclerotic lesions and have been shown to stimulate MMP-1 expression in fibroblasts, smooth muscle cells, and other neoplastic tissues.² However, a recent in vitro study¹⁴ showing that TNF-, IL-1ß, and interferon- IFN- had no effect on MMP-1 expression in human monocyte-derived macrophages did not support this hypothesis. Thus, evidence suggests that local factors other than cytokines may stimulate MMP-1 expression in lesion-associated macrophages.

It has been previously demonstrated that atherosclerotic lesions contain large quantities of immune complexes (ICs), including LDL-containing ICs (LDL-ICs).^{15 16 17} In the present in vitro study, we investigated the roles of LDL-ICs and other ICs in MMP-1 expression and secretion by human U937 histiocytes (macrophages). We found that cocross-linking of Fc- receptors I and II (FcRI, FcRII) by LDL-ICs induced MMP-1 expression and secretion. We also found that induction of MMP-1 expression was mediated by a protein kinase C (PKC)–dependent mitogen-activated protein kinase (MAPK) signaling pathway. Thus, this study demonstrates for the first time that interaction of ICs with macrophages stimulates MMP-1 expression and secretion.

	Methods

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Cell Culture Experiments
U937 histiocytes¹⁸ were cultured in a 5% CO₂ atmosphere in Iscove’s modified Dulbecco’s media supplemented with 10% fetal calf serum. The medium was changed every 2 to 3 days. The histiocytic (resident macrophage) origin of U937 cells was shown by their capacity for lysozyme production and the strong esterase activity.¹⁸ Peripheral blood human monocytes were isolated from plasma collected from healthy, normolipidemic human volunteers as previously described¹⁹ by using the method of Recalde,²⁰ with modifications described by Fogelman et al.²¹

Isolation of Lipoproteins and Preparation of Insoluble ICs
LDL (d=1.019 to 1.063 g/mL) was isolated from the plasma of normal volunteers and oxidatively modified as described.²² Insoluble LDL-ICs were prepared with human native LDL and rabbit anti-LDL antiserum and quantified as described previously.^{23 24 25} Our previous studies had shown that LDL-ICs prepared with both human LDL and rabbit anti-LDL antiserum and those prepared with collagen I–immobilized human oxidized LDL (oxLDL) and anti–oxLDL autoantibodies activated MAPK in macrophages,²² suggesting that the former can be used as a model for the latter in studies examining the interaction of LDL-ICs with macrophages.

ELISA of Secreted MMPs
Secreted MMPs from U937 cells were quantified by using sandwich ELISA kits according to the protocol provided by the manufacturer (Oncogene).

Northern Blot Analysis
Total cellular RNA was isolated from U937 cells by using the Ultraspec RNA isolation reagent according to the instructions from the manufacturer (Biotecx Laboratories). Northern blotting of MMP-1 mRNA was performed as described previously.²⁶

FcR Blocking
FcRI was blocked with human monomeric IgG₁ isolated from serum by NH₄(SO₄)₂ precipitation followed by affinity chromatography on a protein A/G column (ImmunoPure, Pierce) as described previously.¹⁹ Human monomeric IgG₂ was used as negative control for IgG₁.^{19 27} Analysis of IgG subclasses by radial immunodiffusion (Binding Site) on the purified IgG₁ showed mild contamination with IgG₂ (2.4% of the total IgG). To eliminate aggregates, purified IgG₁ and IgG₂ were centrifuged at 100 000g for 30 minutes before being added to the culture medium. FcRII was blocked with a monoclonal anti-CD32 antibody (composition, IgG2b,, clone FLI8.26; PharMingen). A monoclonal antibody (clone 27-35) with the same isotype (IgG2b,) was used as a negative control for anti-CD32.

MAPK Phosphorylation
Phosphorylation of MAPK was detected by Western blot analysis by using monoclonal anti-phosphorylated and anti-p42/p44 MAPK antibodies (Santa Cruz Biotechnology) as described previously.²²

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

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

Statistical Analysis
Data are presented as mean±SEM. Comparison between treatments was performed by using a 1-way ANOVA. A value of P<0.05 was considered significant.

	Results

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Concentration- and Time-Dependent Stimulation of MMP-1 Secretion by LDL-ICs
U937 cells were treated with increasing concentrations (0 to 200 µg/mL) of LDL-ICs for 24 hours, and the amount of secreted MMP-1 in conditioned medium was assayed by ELISA. Results showed that MMP-1 secretion was LDL-IC concentration–dependent and reached a plateau at 150 µg/mL of LDL-ICs (please see Figure I, published online at http://atvb.ahajournals.org). The kinetic study on MMP-1 secretion in response to LDL-ICs showed that MMP-1 secretion was time-dependent and started to plateau after 24-hour stimulation (please see Figure II, published online at http://atvb.ahajournals.org). On the basis of these results, we chose 150 µg/mL and 24 hours as the optimal concentration of LDL-IC and the stimulation time, respectively, for the remaining experiments.

Effects of LDL-ICs on Secretion of MMPs and TIMP-1
The amount of MMP-1, -2, -3, and -9 secreted by U937 cells in response to LDL-ICs was measured by ELISA. Our results showed that LDL-ICs stimulated the secretion of MMP-1 by 20-fold (Figure 1A). In contrast, LDL-ICs inhibited MMP-2 secretion by 50% and had no effect on the secretion of MMP-3 and -9 (data not shown). Our data also showed that both native and oxLDL had no effect on the secretion of MMP-1 (Figure 1A) or MMP-2 (data not shown). Phorbol-12-myristate-13-acetate (PMA) as a positive control induced marked secretion of MMP-1 (Figure 1A). These results demonstrated that LDL-ICs selectively stimulated MMP-1 secretion by U937 cells. We also investigated whether or not the stimulation of MMP-1 was accompanied by an increase in the secretion of tissue inhibitor of metalloproteinase-1 (TIMP-1). Data showed that TIMP-1 secretion was not induced by LDL-ICs (Figure 1B) but was inhibited by oxLDL. The inhibition of TIMP-1 by oxLDL in macrophages was also recently reported by Shah and coworkers.²⁸ In contrast, a 4-fold increase in TIMP-1 secretion was observed in cells stimulated with PMA (Figure 1B).

View larger version (20K): [in this window] [in a new window]   Figure 1. . Effects of LDL-ICs on secretion of MMP-1 and TIMP-1 by U937 cells. U937 cells were incubated at 37°C for 24 hours with culture medium alone (control) or with medium containing 150 µg/mL LDL-IC, 100 µg/mL native LDL, 100 µg/mL oxLDL, 50 µL/mL anti-LDL antiserum, or 100 nmol/L PMA. After incubation, secreted MMP-1 (A) and TIMP-1 (B) in the conditioned medium were quantified by ELISA, and the cells were lysed for DNA assay as described in Methods. Data represent the mean±SEM of 3 experiments run in duplicate.

Induction of MMP-1 Expression in U937 Cells by LDL-ICs
The effect of LDL-ICs on the steady-state level of MMP-1 mRNA in U937 cells was determined by Northern blotting. As shown in Figure 2, MMP-1 mRNA was undetected in control cells but induced by LDL-ICs. PMA, which has been shown to stimulate MMP-1 transcription in human monocyte-derived macrophages,²⁹ markedly increased the MMP-1 mRNA level. These results suggest that stimulation of MMP-1 secretion by LDL-ICs is most likely secondary to an increase in the MMP-1 mRNA level.

View larger version (71K): [in this window] [in a new window]   Figure 2. . Northern blot of MMP-1 mRNA expression in U937 cells stimulated with LDL-ICs. U937 cells were incubated for 24 hours with medium alone (C) or with medium containing 100 nmol/L PMA (P) or 150 µg/mL LDL-ICs (IC). After incubation, total RNA was isolated and 20 µg of RNA for each sample was used for Northern blot analysis of MMP-1 and GAPDH mRNA as described in Methods. The experiment was performed with duplicate dishes for control and LDL-IC–stimulated cells.

Stimulation of Collagenase Activity by LDL-ICs
Collagenase activity in cell-conditioned medium was determined bu using fluorescein-conjugated type I collagen as a substrate. Our results showed that collagenase activity in medium conditioned by cells exposed to LDL-ICs was significantly higher than that observed with medium collected from control cells (data not shown). Our data also showed that the collagenase activity stimulated by LDL-ICs was completely inhibited by 1 mmol/L 1,10-phenanthroline (data not shown), indicating that the collagenase activity was secondary to the presence of metalloproteinases.

Engagement of FcRI and FcRII by LDL-ICs Is Required for Induction of MMP-1 Secretion
Our previous studies have shown that LDL-ICs engage FcRI predominantly and engage FcRII to a lesser extent in human macrophages and THP-1 macrophage-like cells.¹⁹ To determine whether MMP-1 secretion and expression induced by LDL-ICs in U937 cells was due to the engagement of FcRI or FcRII by LDL-ICs, we conducted experiments in which incubation of the cells with LDL-ICs was performed in the presence of FcRI or FcRII blockers. Human monomeric IgG₁, which binds to FcRI with high affinity, and the monoclonal anti-CD32 (FcRII) antibodies (clone FLI8.26) were used to block the interactions of LDL-ICs with FcRI and FcRII, respectively. Our results showed that human monomeric IgG₁ and anti-CD32 inhibited LDL-IC–stimulated MMP-1 secretion in a dose-dependent manner, with 70% and 55% inhibition at 20 and 5 µg/mL, respectively (Figure 3). Human monomeric IgG₂ was used as a negative control, because it does not block either FcRI or FcRII. As expected, human monomeric IgG₂ showed no effect on blocking the LDL-IC–stimulated MMP-1 secretion. Mouse monoclonal antibody clone 27-35, an isotype control for anti-CD32 (IgG2b,), also had no effect on MMP-1 secretion induced by LDL-ICs (Figure 3). Because U937 cells lack FcRIII,³⁰ anti-CD16 (FcRIII) antibody was also used as an irrelevant antibody to exclude the nonspecific interaction between antibodies and U937 cells. Our results showed that anti-CD16 did not block MMP-1 secretion (Figure 3). These data strongly suggest that cocross-linking of FcRI and FcRII by LDL-ICs might be responsible for the induction of MMP-1 secretion in U937 cells.

View larger version (19K): [in this window] [in a new window]   Figure 3. . Blocking of LDL-IC–stimulated MMP-1 secretion by monomeric human IgG1 and anti-CD32 monoclonal antibodies. U937 cells were incubated for 24 hours with 150 µg/mL LDL-ICs in the absence or presence of increasing concentrations of human IgG1, IgG2, anti-CD32 monoclonal antibodies, an anti-CD32 isotype control antibody (mouse IgG2b,), or anti-CD16 monoclonal antibodies as indicated. After incubation, the conditioned medium was subjected to ELISA to determine the amount of secreted MMP-1. The amount of LDL-IC–stimulated MMP-1 in the absence of blocking antibodies was designated as 100%. Data presented are the means of 3 experiments run in triplicate.

LDL-ICs Stimulate MMP-1 Secretion via Activation of a PKC-Dependent MAPK Pathway
Cross-linking of FcRI triggers activation of MAPK pathways.³¹ Our recent study has demonstrated that LDL-ICs activate MAPK (extracellular signal–related protein kinase [ERK]) in THP-1 macrophage-like cells through engaging FcRI.²² On the basis on these observations, we determined whether the altered MMP-1 expression/secretion had resulted from activation of an MAPK pathway induced by LDL-ICs. First, we examined the effect of LDL-ICs on phosphorylation of p42/p44 MAPK (ERK1/2) in U937 cells by Western blot analysis with the use of both anti-phosphorylated and anti–total ERK antibodies. Our data showed that LDL-ICs induced phosphorylation of ERK, mainly ERK2, in a time-dependent manner, with peak phosphorylation occurring at 30 minutes (Figure 4). We then performed experiments in which U937 cells were treated with LDL-ICs in the absence or presence of PD98059,³² and the amount of MMP-1 released into the culture medium was determined after treatment. Our results showed that PD98059 inhibited MMP-1 secretion in a dose-dependent manner, with complete inhibition at 50 µmol/L (Figure 5). Dimethyl sulfoxide (DMSO), a vehicle for PD98059, had no blocking effect on MMP-1 secretion. To ensure that PD98059 inhibited ERK phosphorylation in U937 cells, Western blotting was performed and the results showed that 50 µmol/L PD98059 significantly inhibited ERK phosphorylation (data not shown).

View larger version (38K): [in this window] [in a new window]   Figure 4. . Time-dependent stimulation of MAPK in U937 cells by LDL-ICs. U937 cells were stimulated with 150 µg/mL LDL-ICs for the times indicated and then lysed. Twenty-five micrograms of cell protein was electrophoresed on a 10% SDS polyacrylamide gel and then transferred to a polyvinylidene difluoride membrane. The membrane was immunoblotted with anti-phosphorylated or anti-p42/p44 MAPK antibodies as described in Methods. MAPK was visualized by incubating the membrane with chemiluminescence reagent for 1 minute and exposure to x-ray film for 15 seconds. Data are representative of 3 experiments with similar results.

View larger version (18K): [in this window] [in a new window]   Figure 5. . Inhibition of LDL-IC–stimulated MMP-1 secretion by PD98059. U937 cells were incubated for 24 hours with 150 µg/mL LDL-ICs in the presence of increasing concentrations of PD98059 as indicated. After incubation, the conditioned medium was subjected to ELISA to quantify secreted MMP-1. The amount of secreted MMP-1 by LDL-IC–stimulated cells in the absence of PD98059 was designated as 100%. Dimethyl sulfoxide (DMSO), a vehicle for PD98059, was 0.1% of the medium volume. Data presented are the mean±SEM of 3 different experiments run in duplicate.

Because our recent study²² showed that activation of the MAPK signaling pathway in macrophage by LDL-ICs was PKC dependent, the role of PKC in MAPK-mediated MMP-1 secretion was also investigated. Our results showed that the PKC inhibitor Ro-31-8220³³ blocked MMP-1 secretion in a concentration-dependent manner, and complete inhibition was achieved at 80 nmol/L (please see Figure III, published online at http://atvb.ahajournals.org). The effect of Ro-31-8220 on ERK phosphorylation was determined, and the results showed that 80 nmol/L Ro-31-8220 significantly inhibited ERK phosphorylation (please see Figure IV, published online at http://atvb.ahajournals.org). These data suggest that LDL-ICs induced MMP-1 secretion via a PKC-dependent MAPK signaling pathway.

Stimulation of MMP-1 Secretion by IgG–Anti-IgG ICs
Because atherosclerotic lesions also contain ICs other than LDL-ICs,¹⁷ we determined whether these non-LDL ICs also stimulated MMP-1 secretion by U937 cells. Insoluble IgG–anti-IgG ICs IgG-ICs were prepared by incubating human IgG with rabbit anti-human IgG antiserum. Our results showed that insoluble IgG–anti-IgG ICs also stimulated MMP-1 secretion by U937 cells (please see Figure V, published online at http://atvb.ahajournals.org). Thus, these data indicate that insoluble IgG-containing ICs are capable of inducing MMP-1 secretion by U937 cells, regardless of their antigen content.

Stimulation of MMP-1 Secretion From Human Monocyte-Derived Macrophages by LDL-ICs
We further determined whether LDL-ICs also stimulated MMP-1 secretion by human monocyte-derived macrophages. Our results showed that treatment of macrophages with LDL-ICs resulted in a 3-fold increase in MMP-1 secretion over control levels (please see Figure VI, published online at http://atvb.ahajournals.org).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The titer of anti-oxLDL autoantibodies has been measured in human serum by our and other laboratories.^{34 35 36 37 38} In addition to serum, the unbound autoantibodies as well as those complexed with oxLDL have been also localized in atherosclerotic lesions.¹⁷ The potential role of LDL-ICs in atherogenesis has been extensively investigated by our and other groups (for a review, see Reference 38). In our previous studies of insoluble LDL-ICs prepared with human LDL and rabbit anti-LDL antiserum, we demonstrated that interaction between LDL-ICs and macrophages led to foam cell formation and macrophage activation.^{24 39} Recently, we have demonstrated that this interaction was mediated predominantly by FcRI.¹⁹ Our present study shows that MAPK activation elicited by interaction between LDL-ICs and the FcRs on U937 histiocytes led to induction of MMP-1 expression and secretion. Because MMP-1 has been shown to be potentially involved in the disruption of atherosclerotic plaques,² our present study suggests that LDL-ICs may play an important role not only in the initiation and progression of atherosclerosis but also in triggering acute coronary events.

TIMP-1 and MMP-1 have been shown to be coordinately regulated by some factors, such as PMA and interleukin-1ß, and reciprocally regulated by others, such as transforming growth factor-ß1, which downregulates TIMP-1 while upregulating MMPs.⁴⁰ Our present study has shown that LDL-ICs stimulate MMP-1 secretion but have no effect on TIMP-1, resulting in a net increase in collagenase activity. The complete inhibition of stimulated collagenase activity by phenanthroline indicates that the enhanced collagenase activity is secondary to the increased secretion of MMPs. Because phenanthroline is not a specific inhibitor of MMP-1, our data do not ascertain that MMP-1 is the responsible enzyme. Although MMP-1 might be at least partially responsible for increased collagenase activity, because our results show that LDL-ICs selectively stimulate MMP-1 and the fluorescence-labeled type I collagen is the substrate of MMP-1, the involvement of MMP-13 needs to be excluded. Libby and coworkers⁴¹ recently demonstrated increased levels of MMP-13 and MMP-1 and the loss of interstitial collagen type I in atheromatous versus fibrous plaques.

An intriguing result of this study is that cocross-linking of FcRI and FcRII by LDL-ICs seems to be essential for induction of MMP-1 secretion, because human monomeric IgG₁ and anti-CD32 monoclonal antibody blocked the MMP-1 secretion induced by LDL-ICs by 70% and 55%, respectively. To the best of our knowledge, this is the first report to show that cocross-linking of FcRI and FcRII coordinates gene expression. Because ICs are capable of binding either FcRI or FcRII on the surface of U937 cells, they can either cocross-link FcRI and FcRII separately and simultaneously or cocross-link FcRI with FcRII. Our blocking study did not distinguish the cocross-linking that plays an essential role in MMP-1 stimulation, and further studies are required to address this issue. Previously, a number of studies on lymphocytes have reported that cocross-linking of 2 different cell surface receptors by ligands is needed for regulating specific cell functions.^{42 43 44} For example, it has been shown that in B cells, cocross-linking of Fc-RII and the B-cell receptor modulates B-cell activation⁴² and that cocross-linking of CD27 and the B-cell receptor augments CD27-mediated B-cell apoptosis.⁴³ In T cells, it has been shown that cocross-linking of CD3 and CD4 results in enhanced mobilization of free intracellular calcium.⁴⁴ Our previous studies on human monocyte-derived macrophages showed that FcRI was engaged by LDL-ICs predominantly and that FcRII was engaged to a lesser extent.¹⁹ Despite the fact that FcRII is less engaged by LDL-ICs, our present study showed that 55% of MMP-1 secretion by U937 cells was inhibited by blocking FcRII, suggesting that FcRII may play an important role in MMP-1 expression.

Unlike FcRI as a single form, FcRII has several isoforms: FcRIIA, IIB1, IIB2, and IIC.⁴⁵ It has been shown that FcRIIB molecules are preferentially expressed by lymphocytes, whereas FcRIIA and IIC are preferentially expressed by neutrophils, and that human monocytes and macrophages express all classes.⁴⁵ However, the surface expression of these isoforms on human monocytes/macrophages remains unknown, because the extracellular and transmembrane domains of these isoforms are nearly identical and the monoclonal antibodies that distinguish among the surface epitopes of FcRIIA, IIB, and IIC have not been successfully produced.⁴⁵

In contrast to the extracellular and transmembrane domains, the cytoplasmic portions of FcRII isoforms are divergent.³¹ FcRIIA/C contains an immunoreceptor tyrosine-based activation motif, whereas FcRIIB1/B2 contains an immunoreceptor tyrosine-based inhibition motif (ITIM).³¹ Owing to the complexity of the surface expression of FcRII isoforms on monocytes/macrophages, the role of the ITIM-containing FcRIIB in monocyte/macrophage activation remains unknown. However, many studies have shown that cross-linking of FcRII on monocytes/macrophages led to activation of signaling pathways,^{30 46 47} suggesting that the inhibitory signal transmitted through ITIM-containing FcRIIB may not mediate a complete shutdown of FcRIIA/C-mediated tyrosine phosphorylation in human monocytes. Further extensive investigation is necessary to define the role of FcRIIB in signal transduction and gene expression in monocytes and macrophages.

In summary, our present study demonstrates for the first time that cocross-linking of FcRI and FcRII by LDL-ICs induces MMP-1 expression and secretion by U937 histiocytes via activation of a PKC-dependent MAPK signaling pathway. These results suggest that the interaction between ICs and macrophages in atherosclerotic plaques may lead to induction of MMP-1 secretion, thus contributing to the disruption of atherosclerotic plaques and acute coronary events.

	Acknowledgments

 
This work was supported by an institutional grant from the Medical University of South Carolina, the Atorvastatin Research Award, a Merit Review Grant from the Research Service of the Department of Veterans Affairs (to Y.H.), and in part by grant HL-55782 from the National Institutes of Health (to M.F.L.-V.). We thank Charlyne Chassereau for the preparation of lipoproteins.

	Footnotes

 
Part of this work was presented at the American Heart Association 72nd Scientific Sessions and was published in abstract form (Circulation 1999;[suppl I]100:I-252).

Received August 3, 2000; accepted September 6, 2000.

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