Stabilization of Atherosclerotic Plaques by Blockade of Macrophage Migration Inhibitory Factor After Vascular Injury in Apolipoprotein E–Deficient Mice
Background— Macrophage migration inhibitory factor (MIF), a cytokine that controls cell-mediated inflammatory responses, is upregulated in atherogenesis; however, its functional contribution to lesion development has not been evaluated.
Methods and Results— We studied the role of MIF on neointima lesion formation after wire-induced injury of carotid arteries in apolipoprotein E–deficient (apoE−/−) mice. Immunohistochemistry revealed that MIF expression was detectable in endothelial cells before injury and upregulated in smooth muscle cells (SMCs) 24 hours after endothelial denudation. Three weeks after injury, MIF was predominantly found in endothelial cells and macrophage-derived foam cells. Neutralizing MIF with a monoclonal antibody resulted in a marked reduction of neointimal macrophages and inhibited transformation of macrophages into foam cells. Conversely, the content of SMCs and of collagen in the neointima were increased, amounting to a slight but not significant reduction in neointima and media size after 3 weeks of MIF monoclonal antibody treatment. Notably, serum levels of the cytokines IL-2, IL-4, IL-6, IL-10, and tumor necrosis factor were increased in MIF monoclonal antibody–treated mice. In vitro flow assays revealed that MIF pretreatment of aortic endothelium enhanced monocyte recruitment and that the monocyte arrest induced by oxidized LDL is mediated by endothelial MIF, as shown by monoclonal antibody inhibition.
Conclusions— Inhibition of MIF resulted in a shift in the cellular composition of neointimal plaques toward a stabilized phenotype with reduced macrophage/foam cell content and increased SMC content. This might be attributable to a reduction of monocyte recruitment mediated by endothelial MIF.
Received June 3, 2003; revision received September 11, 2003; accepted September 18, 2003.
Restenosis caused by accelerated neointima formation is a frequent long-term complication after percutaneous transluminal coronary angioplasty in patients with obstructive coronary atherosclerosis. Phenotypically modified smooth muscle cells (SMCs) constitute a major part of restenotic lesions.1 In addition, macrophages and macrophage-derived foam cells significantly contribute to the neointimal tissue and further aggravate the local inflammatory response and neointimal growth by expression of cytokines and growth factors, especially in the context of hypercholesterolemia.2–4 After endothelial denudation and apoptosis of medial SMCs, leukocyte recruitment by densely aggregated platelets and proliferation of SMCs occur at the injury site.1,5 Ongoing monocyte recruitment during neointima formation is supported by neointimal SMCs or activated endothelium, depending on the status of reendothelialization.6 Furthermore, active inflammation based on infiltration with macrophages and T cells promotes plaque instability by weakening of the fibrous cap and facilitating plaque disruption and subsequent thrombosis.7
Macrophage migration inhibitory factor (MIF) is a pleiotropic macrophage and T-cell cytokine, endocrine factor, and enzyme. MIF was found to be crucial in regulating immune-mediated diseases8 such as chronic colitis,9 septic shock,10,11 arthritis,12 and delayed-type hypersensivity.13 The molecular basis of these functions is still incompletely understood, although the enzymatic activities of MIF, intracellular formation of complexes of MIF with the transcriptional coactivator JAB1,14 and CD74-mediated triggering of the MAPK pathway by MIF have been implicated.15 As to its role in local tissue inflammation, the pathogenicity of MIF has been attributed to monocyte and T-cell recruitment in a rat model of glomerulonephritis.16 Moreover, a role for MIF in atherogenesis has been proposed, since upregulation of MIF has been observed in endothelial cells, SMCs, and macrophages during progression of atherosclerotic plaques in humans17 and in a hypercholesterolemic rabbit model.18 Although a contribution of MIF to macrophage accumulation in atherosclerotic lesions has been postulated, the precise function of MIF in atherogenesis remains unclear.
Our data reveal that inhibition of MIF supports a stabilization of neointimal plaques with reduced macrophage/foam cell and increased SMC content. This appears to be due to a reduction of monocyte recruitment mediated by endothelial MIF.
Atherogenic Mouse Model of Restenosis
Female 8-week-old apoE−/− mice (C57BL/6, M&B, Ry, Denmark) were fed an atherogenic diet containing 21% fat for 1 week before and up to 3 weeks after injury. Transluminal wire injury of the left common carotid arteries was performed as reported.19 In brief, mice were anesthetized with intraperitoneal ketamine/xylazine; a 0.014-inch flexible angioplasty guide wire was advanced 1 cm through transverse arteriotomy of the external carotid artery, and endothelial denudation was achieved by 3 rotational passes along the common carotid artery. Treatment with MIF monoclonal antibody (mAb) (clone III D.9, derived from the Bucala Laboratory as described,20 100 μg IP twice weekly) or isotype control (n=5 per group) was started 12 hours before wire injury. The mAbs were prepared by HiTrapQ FPLC affinity chromatography. The lipopolysaccharide (LPS) concentration was <0.05 fg/ng protein as determined by Limulus amoebocyte assay. After 3 weeks, the left carotid artery was excised after in situ perfusion-fixation with 4% paraformaldehyde and paraffin-embedded. Animal experiments were approved by the local authorities and complied with German animal protection law. Total cholesterol and triglyceride serum levels were determined with standard enzymatic assays (Roche).
Serial tissue sections (5 μm) were obtained from the left common carotid arteries, starting at the bifurcation. Ten sections (50 μm apart) were stained according to Movats modified pentachrome.18 Areas within lumen, internal and external elastic laminae, were measured by planimetry of digitized images obtained by a bright-field microscope (Leica DMLB) with the use of Diskus software (Hilgers), and neointimal and medial plaque volumes were calculated as area under the curve.
Immunohistochemistry and Double Immunofluorescence Staining for MIF
Carotid sections from hypercholesterolemic apoE−/− mice either uninjured or 24 hours, 2 weeks, and 3 weeks after wire injury were prepared for immunostaining by antigen retrieval through the use of a citrate buffer (pH 6) in a microwave oven and subsequently by blocking nonspecific background with 5% horse serum and an avidin-blocking agent (Vector Laboratories). Slides were incubated with goat polyclonal MIF antibody (2 μg/mL, Santa Cruz) or control antibody, 5% normal horse serum, and biotin at 4°C overnight; reacted with biotinylated secondary antibody, avidin-biotin alkaline phosphatase complex, and Vector Red substrate; and counterstained with hematoxylin.
To identify cellular MIF immunoreactivity (IR), double-immunofluorescence staining of carotid sections 3 weeks after injury was performed. MIF immunostaining with the fluorescent Vector Red product was combined with staining for α-smooth muscle actin (α-SMA, clone 1A4, Dako), Mac-2 (clone M3/38, Cedarlane) visualized with a FITC-conjugated secondary antibody or VE-cadherin (polyclonal antibody, Santa Cruz) detected with a HRP-conjugated secondary antibody followed by tyramide amplification (Perkin Elmer), and a streptavidin-FITC conjugate (Vector).
Quantitative Immunohistochemistry and Immunofluorescence
Sections (3 to 4 per mouse, 50 μm apart) from neointimal lesions of mice treated with MIF mAb or isotype control (n=5 per group) were immunostained for cellular differentiation markers and collagen. Macrophages or SMCs were identified by using antibody against α-SMA, Mac-2, or respective isotype controls. Specific binding was detected with a biotinylated-secondary antibody, avidin-biotin peroxidase complex, and 3,3′-diaminobenzidine substrate (Vector Labs). Other slides were incubated with polyclonal collagen type I antibody (Cedarlane) followed by an FITC-labeled antibody. Digital images were analyzed for total neointimal area and the area with specific immunostaining through the use of Analysis Software (Soft Imaging Systems). Data for each cell type and collagen were expressed as percentage of the immunostained area per total neointimal area. Foam cell index was calculated from the percentage of foam cell area per total neointimal area measured in Movats pentachrome–stained sections divided by the percentage of neointimal Mac-2–positive area, including macrophage-derived foam cells, in two consecutive sections.
ELISA for Cytokines and MIF mAb
Mouse serum cytokines were measured after 3 weeks of antibody treatment (n=5 per group) with the use of SearchLight Mouse Cytokine Array (PerbioScience). To determine MIF mAb levels in the mice, a direct nonsandwich ELISA was developed. Briefly, plasma samples were incubated in 96-well plates coated with recombinant mouse MIF21 (500 ng/mL) and reacted with biotinylated secondary antibody, streptavidin-HRP, and tetramethyl benzidine peroxidase substrate (Sigma). Absorbance was read at 450 nm, background was corrected, and MIF mAb concentrations were calculated by using clone III D.9 mAbs as standards.
Monocyte Adhesion on Human Aortic Endothelial Cells in Shear Flow
Laminar flow assays were performed as described.19 Briefly, confluent human aortic endothelial cells (HAoEC, PromoCell) grown in Petri dishes with endothelial growth medium MV (PromoCell) were preincubated with or without 50 ng/mL recombinant human MIF (rhMIF) for 2 hours at 37°C or control buffer and coincubated with blocking MIF mAb (10 μg/mL) or isotype control. rhMIF was prepared by bacterial expression and chromatographic purification as described20 and was biologically active and free of endotoxin (<10 fg LPS/ng MIF). Oxidized LDL (oxLDL) or native LDL-pretreated (each 10 μg/mL, Paesel & Lorei) HAoECs (24 hours, 37°C) were coincubated with either MIF mAb (20 μg/mL) or isotype control. MonoMac6 cells (106/mL) in assay buffer (10 mmol/L HEPES, 1 mmol/L Ca2+/Mg2+, and 0.5% bovine serum albumin) were perfused at 1.5 dyne/cm2. Firm adhesion after 2 minutes was analyzed in multiple high-power fields, recorded by videomicroscopy.
Western Blot and ELISA Analysis of Serum MIF
For Western analysis of serum MIF, electrophoresis and immunoblotting on nitrocellulose membranes was performed essentially as described.11 Briefly, 5 μL of mouse serum aliquots were separated in 4% to 12% NuPAGE gels, with the use of MES buffer. Proteins were blotted, and serum MIF revealed with a polyclonal MIF Ab14 (Ka 565) in combination with peroxidase-labeled secondary antibody and enhanced chemiluminescence. ELISA for mouse MIF was performed as described,10 using MIF mAb (clone XIV D.3) as capture antibody.
Data are expressed as mean±SEM. Statistical analysis was performed with Prism 4 software (Graph Pad) by use of 2-tailed Student’s t test and Welch’s correction if appropriate or 1-way ANOVA with Newman-Keuls post test. Differences with a value of P<0.05 were considered statistically significant.
MIF Expression After Vascular Injury
Expression of MIF has been found in various cell types of atherosclerotic plaques in rabbits and humans.17,18 Since neointima formation after vascular injury resembles an accelerated form of atherosclerosis, we evaluated MIF expression in carotid lesions of apoE−/− mice after wire-induced injury by immunohistochemistry. In uninjured carotid arteries of hypercholesterolemic apoE−/− mice, MIF-IR was exclusively and specifically detected in endothelial cells (Figure 1, A and B). In injured carotid arteries, MIF-specific staining was also found in medial SMCs next to the denuded area after 24 hours (Figure 1C). A more prominent staining for MIF was observed 2 weeks after injury in foam cells and in luminal cells (Figure 1D). Combined immunofluorescence staining for MIF and cell type-specific markers confirmed MIF expression by macrophages (Figure 2A), macrophage-derived foam cells (Figure 2A), and endothelial cells (Figure 2B), but only in a small subset of neointimal SMCs (data not shown).
Circulating MIF levels determined by immunoblotting and ELISA analysis were strongly increased in 2 of 5 mice 6 hours after wire injury as compared with preinjury levels, whereas none of the mice revealed changes in MIF levels at 24 and 48 hours (data not shown).
Effect of MIF Blockade on Plaque Size
To study the functional role of MIF in neointima formation, apoE−/− mice were treated with a neutralizing MIF mAb or an isotype control for 3 weeks. Administration of the MIF mAb resulted in plasma levels of 219±28 μg/mL at 24 hours and of 98±24 μg/mL at 96 hours after injection (n=3) and were thus in large excess of the dose needed to neutralize MIF in vitro. Body weight, total cholesterol, and triglyceride levels did not differ between MIF mAb–treated and isotype control–treated mice (Table). Although neointimal and medial volumes were slightly reduced in MIF mAb–treated mice, these differences were not significant (Table).
Plaque Composition After MIF Blockade
Next, we examined the effect of MIF blockade on cellular composition and collagen content in neointimal lesions by immunostaining. In the MIF mAb–treated mice, Mac-2–positive macrophages were markedly reduced in neointimal lesions (52.3±6.4% versus 26.2±6.3% area, n=5, P<0.02) (Figure 3, A through C). To study the role of MIF in foam cell development, the proportion of foam cells among total neointimal Mac-2–positive macrophages (foam cell index) was determined. In isotype control-treated apoE−/− mice, 85.8±1.8% of Mac-2–positive macrophages were identified as foam cells. Treatment with the neutralizing MIF mAb significantly reduced the percentage of foam cells among Mac-2–positive cells by 51.6% (Figure 3D). Conversely, neointimal immunostaining for α-SMA was significantly increased (22.7±1.5% versus 44.2±4.0% area, n=5, P<0.001) in these mice (Figure 4A). This was paralleled by a marked increase of neointimal collagen type I content in MIF mAb–treated mice (16.4±2.6% versus 28.0±4.0% area, n=5, P<0.05) (Figure 4B).
MIF-Dependent Changes in Serum Cytokine Concentrations
MIF affects the synthesis and secretion of various cytokines in systemic disease models, such as endotoxemia, which may account for part of its pathogenetic activity.11,22 Since MIF blockade may influence cytokine production in apoE−/− mice after vascular injury, the serum levels of various cytokines were determined in MIF mAb–treated and isotype control–treated mice. Three weeks after wire-induced carotid injury, serum levels of IL-4, IL-2, IL-6, IL-10, and TNF were significantly increased in MIF mAb–treated mice, whereas IL-1β serum levels were slightly higher in isotype control–treated mice and serum levels of IL-5, IL-12, and interferon-γ were found to be unaltered by MIF mAb treatment (Figure 5).
MIF Effect on Monocyte Adhesion in Flow
Since the reduction in neointimal macrophage content caused by the blockade of MIF could be due to either diminished recruitment of monocytes or to a redistribution of neointimal macrophages into the blood, we evaluated the propensity of MIF to regulate monocyte arrest under flow conditions. Pretreatment of HAoEC with rhMIF (50 ng/mL) for 2 hours resulted in a marked increase in monocyte arrest, as compared with control buffer–treated or untreated endothelium (Figure 6A). MIF-triggered arrest was inhibited by coincubation with the blocking MIF mAb (Figure 6A). Oxidized LDL has been reported to induce endothelial MIF expression17; therefore, we studied the contribution of endogenous MIF to monocyte recruitment on oxLDL-treated HAoEC. As compared with isotype control, the blocking MIF mAb diminished monocyte arrest on oxLDL-treated HAoEC by 40% (Figure 6B). Taken together, these data clearly demonstrate the involvement of endothelial MIF in monocyte arrest on endothelium exposed to atherogenic stimulation.
This study demonstrates that immunoneutralization of MIF significantly reduces neointimal macrophage infiltration and macrophage derived foam cell formation in a hypercholesterolemic model of vascular injury. Total plaque size, however, was not significantly changed by MIF blockade, probably as the result of the marked increase in neointimal SMC and collagen type I content. Moreover, our data establish an involvement of endothelial MIF in monocyte arrest in shear flow. The inhibition of MIF-dependent monocyte recruitment may thus be an important mechanism for plaque stabilization.
In hypercholesterolemic but uninjured apoE−/− mice, MIF is expressed in arterial endothelial cells at lesion-prone sites without apparent atherosclerotic plaques. During the course of neointima formation after wire injury, MIF expression is evident in most cell types, especially in the endothelium and in macrophages. Similar findings revealing an upregulation of MIF on different cell types of the plaque have been reported for spontaneous atherosclerosis in humans17 and cholesterol-fed rabbits.18 Moreover, MIF expression was demonstrated in different stages of human atherosclerotic plaque development.17 Hence, our current data indicate a similarity in the cellular pattern of MIF expression in wire-induced neointimal lesions of apoE−/− mice and spontaneous atherosclerosis. Preceding the development of manifest plaques, endothelial cells express MIF at very early stages of native lesion formation in hypercholesterolemic apoE−/− mice, implicating MIF in the initiation of atherogenesis.
The inhibition of the random migration of macrophages has been described as one of the first activities of MIF and is eponymous for the 12.5-kDa protein.8 MIF enhances proinflammatory functions of macrophages when externally administered21 or endogenously expressed by macrophages.23,24 Additional evidence for an important role of MIF in macrophage-dependent diseases has been provided in a model of nephrotoxic glomerulonephritis, in which a neutralizing MIF antibody reduced disease parameters and leukocyte infiltration.16 Our finding that blocking MIF substantially reduces neointimal macrophage content supports the notion that MIF is centrally involved in macrophage recruitment in atherosclerotic vascular disease. The inhibition of macrophage transformation into foam cells by MIF mAb further indicates that MIF participates in a subsequent step of atherosclerotic plaque development orchestrated by macrophages.25 This regulatory function could be due to reduced uptake of oxidized LDL,26 which is instrumental for foam cell formation.
Interestingly, neointimal plaque size was not significantly diminished by systemic application of MIF mAb, despite the marked reduction of macrophage content. This is attributable to the increase in neointimal SMC and collagen type I content, which almost fully compensated for the effect of MIF mAb on neointimal macrophage content. This is in contrast to findings that a reduction of neointimal macrophage content, for example, through inhibition of MCP-14 or RANTES receptors,19 was associated with diminished neointima after vascular injury and may suggest the existence of MIF-specific counterregulatory mechanisms after MIF mAb application. Notably, serum cytokine levels were altered in the MIF mAb–treated mice, as evident by an increase in proinflammatory cytokines, such as TNF and IL-6, which might contribute to an acceleration of neointima formation. Moreover, endogenously expressed MIF participates in intracellular signaling and regulates the proinflammatory activity of macrophages.14,23 Since the function of intracellular MIF is not primarily targeted by MIF mAb treatment, a complete absence of MIF activity, for example, in MIF−/− mice, might exert different effects on neointimal plaque size. Alternatively, effects of MIF on SMC proliferation or collagen turnover in neointimal lesions may be inhibited by MIF mAb treatment.
To clarify the role of MIF in monocyte recruitment, we performed in vitro assays of monocyte adhesion to endothelial cells in flow. The increase in monocyte arrest on endothelium activated by oxLDL, which has been shown to induce endogenous MIF expression,17 was partially mediated by endothelial MIF. The stimulation of monocyte adhesion by oxLDL involves interactions of Mac-1 with ICAM-1, heparin proteoglycans, as well as yet unidentified ligands or agonists.27 Our data indicate a role for endothelial MIF in oxLDL-stimulated monocyte arrest. This effect could be due to an autocrine feedback loop that activates endothelial cells or due to direct activation of monocytes, since it was abolished by extracellular neutralization of MIF. The observation that a short-term incubation of HAoEC with MIF triggers monocyte arrest under flow conditions supports a model in which MIF directly affects endothelial-monocyte interactions by a novel mechanism resembling the function of immobilized chemokines.
In summary, we have identified MIF as an important regulator of the cellular composition in neointimal lesions of hypercholesterolemic apoE−/− mice after vascular injury. Immunoneutralization of MIF resulted in plaque stabilization by reduction of monocyte/macrophage infiltration and foam cell formation. Therefore, MIF appears to be a promising target for pharmacological therapy of early and advanced atherosclerotic vascular disease.
This study was supported by Deutsche Forschungsgemeinschaft (DFG) grants WE 1913/2–3 and 5–1 (C.W.) as well as DFG grant TP A7 SFB 542 (J.B.) and NIH AI43210 (R.B.). We thank the staff of the vivarium at the University Hospital Aachen for excellent care of the animals and H. Lue and H. Fünfzig for stimulating discussions.
Drs Bernhagen and Bucala are coinventors on a patent describing the use of MIF antibody for the treatment of inflammatory disease.
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