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(Circulation. 1999;99:2310-2316.)
© 1999 American Heart Association, Inc.

Basic Science Reports

Monocyte Chemoattractant Protein-1 but Not Tumor Necrosis Factor- Is Correlated With Monocyte Infiltration in Mouse Lipid Lesions

Jill Reckless, BSc, PhD; Edward M. Rubin, MD, PhD; Judy B. Verstuyft, BA; James C. Metcalfe, MA, PhD; David J. Grainger, MA, PhD

From the Departments of Biochemistry and Medicine, University of Cambridge (UK) (J.R., J.C.M., D.J.G.), and Life Sciences Division, Lawrence Berkeley Laboratory, University of California, Berkeley (E.M.R., J.B.V.).

Correspondence to Jill Reckless, Department of Medicine, University of Cambridge, Addenbrookes Hospital, Box 157, Hills Rd, Cambridge CB2 2QQ UK. E-mail jr219{at}mole.bio.cam.ac.uk

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Apolipoprotein (apo)(a) transgenic mice and C57BL/6 mice fed a high fat diet develop similar-sized lipid lesions, but lesions in apo(a) mice are devoid of macrophages. We used this observation to identify which proinflammatory proteins might be involved in mediating monocyte recruitment during atherogenesis.

Methods and Results—Macrophage-deficient apo(a) transgenic mouse lesions contained similar levels of several different proinflammatory proteins, both adhesion molecules (intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]) and cytokines (tumor necrosis factor- [TNF-] and macrophage inflammatory protein-1 [MIP-1]), similar to the macrophage-rich lesions of C57BL/6 mice.

Conclusions—From this we conclude that ICAM-1, VCAM-1, TNF-, and MIP-1 may all be necessary for vascular monocyte recruitment in vivo, but they cannot be sufficient. Monocyte chemoattractant protein-1 (MCP-1) protein was undetectable in the vessel wall taken from apo(a) transgenic mice fed a high fat diet compared with high expression in mice with lipid lesions (C57BL/6 and apoE knockout mice). Therefore elevated expression of MCP-1 but not TNF-, MIP-1, ICAM-1, or VCAM-1 is correlated with vascular macrophage accumulation. To test the hypothesis that monocyte infiltration during atherogenesis is MCP-1 dependent, it will be necessary to develop specific pharmacological inhibitors of MCP-1 activity.

Key Words: atherosclerosis • monocytes • aorta • lipids • lesion

	Introduction

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The infiltration of monocytes into the vascular wall and their transformation into lipid-laden foam cells characterize early atherogenesis.^{1 2 3} This focal accumulation of lipids, together with smooth muscle cell proliferation and migration, and the synthesis of extracellular matrix in the intima of large arteries result in the formation of an atherosclerotic plaque.^{1 4} The extent to which the plaque is infiltrated with monocytes appears to be an important determinant of plaque stability.^{3 5 6 7} It has been proposed that macrophages secrete an excess of matrix-degrading enzymes over their inhibitors, resulting in conversion of a stable plaque into an unstable plaque that is likely to rupture, resulting in acute myocardial infarction.^{6 7 8 9}

Macrophages and T cells constitute 40% of the total population of cells in the lipid core region of atherosclerotic plaques.^{1 10 11} Their recruitment to the lesion may depend on alterations in the adhesive properties of the endothelial surface.^{12 13 14} Increased endothelial cell permeability and endothelial cell activation are among the earliest changes associated with developing lesions of atherosclerosis.^{12 13 14} Many of the cell adhesion molecules involved in monocyte recruitment are expressed at low or undetectable levels on normal endothelium but are substantially elevated on the endothelium overlaying atherosclerotic lesions.¹⁰ In addition to endothelial cell activation, numerous chemotactic cytokines have also been postulated to be involved in monocyte recruitment. For example, interleukin (IL)-1 and tumor necrosis factor- (TNF-) are direct chemoattractants for human monocytes but additionally induce cytoskeletal changes in the endothelium that result in increased permeability.^{15 16} This increased permeability, together with stimulated expression of adhesion molecules such as E-selectin,^{12 17} plays an important part in the local inflammation mediated by TNF- and IL-1.^{15 16} In addition, a large number of other proinflammatory cytokines, including macrophage inflammatory protein-1 (MIP-1) and monocyte chemoattractant protein-1 (MCP-1), are direct chemoattractants for monocytes.^{18 19 20} Thus alteration in the expression of a wide variety of adhesion molecules and/or cytokines during atherogenesis has been proposed to affect monocyte recruitment and hence modulates both plaque development and stability.

To date, more than 20 inflammation-associated cell adhesion molecules and almost 50 proinflammatory cytokines have been described. A significant number of these have already been shown to be present in human atherosclerotic plaques.^{13 15 16 21 22 23} As a result, it is difficult to determine the relative importance of the various proinflammatory pathways in the monocyte recruitment that occurs during human atherogenesis.²⁴ Since the vascular lipid lesions that form in a wide range of animal models of the disease such as the Watanabe heritable hyperlipidemic rabbit^{25 26 27 28} or the apolipoprotein (apo)E knockout mouse^{29 30} are also richly populated with macrophages, it is plausible that the proinflammatory pathways involved in atherogenic monocyte recruitment are similar in mouse and in humans. Fortuitously, however, one animal model of vascular lipid lesion formation, the apo(a) transgenic mouse,^{31 32} has recently been demonstrated to have lesions devoid of macrophages.²³ Macrophages could not be detected in the vessel wall of apo(a) transgenic mice either with the use of various immunochemical techniques or by electron microscopy²³ (Reckless J, Rubin EM, Verstuyft JB, Metcalfe JC, Grainger DJ. Unpublished data, 1998). We have exploited the fact that apo(a) transgenic mice and the inbred mouse line C57BL/6 develop similar-sized vascular lipid lesions when fed a fat-enriched diet except that the apo(a) transgenic mouse lesions are devoid of macrophages and the C57BL/6 mouse lesions are heavily populated with macrophages.

In this study we have used quantitative immunofluorescence to measure the levels of a variety of adhesion molecules and proinflammatory cytokines in vascular lipid lesions derived from these two different mouse lines as well as from apoE knockout mice, which develop the most severe, macrophage-rich lesions yet described. Any proinflammatory proteins present in the lesions from apo(a) transgenic mice may be necessary for monocyte recruitment, but they cannot be sufficient. In contrast, proinflammatory molecules present in lesions from C57BL/6 mice, and to a greater extent in lesions from apoE knockout mice but absent in lesions from apo(a) transgenic mice, are candidates as the key signal orchestrating monocyte recruitment during vascular lipid lesion formation.

	Methods

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Production of Mice
Mice (all female) from 3 lines of mice of the following types were obtained as previously described: apo(a) transgenics,³¹ apoE knockout mice^{29 30} and C57BL/6 mice.^{33 34 35} Both the apo(a) transgenic mice and apoE knockout mice had been crossed into a C57BL/6 background for at least 5 generations. Mice were screened for the transgenes with the use of the appropriate polymerase chain reaction primers as described in the above references.

Diets of Mice
At 12 weeks of age, the mice from each mouse line were split into 2 groups of 4. One group was fed a normal mouse chow (Purina 5001, Ralston-Purina) containing 4.5% fat and <0.03% cholesterol, whereas the remaining group was fed a diet containing high fat (1.25% cholesterol, 0.5% cholic acid, and 15% fat). Each mouse was fed its respective diet for 12 weeks before it was killed. Water and food were freely available throughout the course of the experiment. At the time the mice were killed, the hearts and aortas were removed from each mouse, embedded, and frozen in the cryostat at -26°C as previously described.³⁶

Immunocytochemistry
Sections from the aortic sinus region were collected according to the strategy of Paigen et al.³⁴ Briefly, for quantitative immunofluorescence, 6-µm sections were placed on slides precoated with poly-L-lysine (0.1%; Sigma Chemical Co) and fixed in ice-cold acetone for 90 seconds. The sections were allowed to air dry and then were stored at -20°C until assayed. Three serial sections for each mouse were used to stain for each protein marker and 2 serial control sections for each mouse. Three images per section were captured and quantified as previously described.^{32 37 38} Lipid lesion formation was analyzed by oil red O staining. For each mouse, 5 sections, each separated by 80 µm, were fixed in 10% buffered formalin, stained with oil red O, and counterstained with light green. The area of oil red O staining in each section was quantified with a calibrated eyepiece. Regions of focal oil red O staining >500 µm² were defined as focal lipid lesions.

Measurement of Macrophages in Aortic Vessel Wall Sections
Two different anti-mouse specific macrophage primary antibodies were used on serial sections from each mouse for this study (each rat IgG2b subclass; Mac-1 and Mas034b). Mac-1 (CD11b/CD18; clone M1/70, Boehringer Mannheim) was used at 2 µg/mL and Mas034b was used at 10 µg/mL (clone M1/7015, SeraLab) for 16 hours at 4°C. Fluorescein (FITC)-labeled anti-rat IgG (Sigma) was used as the secondary antibody at a dilution of 1:20 for 6 hours at room temperature.

Measurement of Adhesion Proteins in Aortic Vessel Wall Sections
Anti-mouse intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) rat monoclonals (IgG2b and IgG1, respectively) were used on serial sections from each mouse for this study. ICAM-1 (clone KAT-1; R and D Systems) and VCAM-1 (clone M/K-2; R and D Systems) were both used at 20 µg/mL for 16 hours at 4°C and room temperature, respectively. Rhodamine (TRITC)-conjugated affinipure mouse anti-rat IgG (code: 212-025-102; Jackson Immunoresearch) was used as the secondary antibody at 30 µg/mL for 6 hours at room temperature.

Measurement of Proinflammatory Cytokines in Aortic Vessel Wall Sections
Anti-mouse TNF-, MIP-1, and JE/MCP-1 polyclonal antibodies (goat IgG) were used on serial sections from each mouse for this study. TNF- (code AB-410-NA, R and D Systems), MIP-1 (code AB-450-NA, R and D Systems), and MCP-1 (code AB-479-NA, R and D Systems) were all used at 50 µg/mL for 16 hours at 4°C. FITC-conjugated affinipure donkey anti-goat IgG (code: 705-095-147; Jackson Immunoresearch) was used as the secondary antibody at 30 µg/mL for 6 hours at room temperature.

Confocal Microscopy
Confocal microscopy was conducted with the use of a Biorad MRC 1000 system interfaced with an argon/krypton ion laser and with fluorescence filters and detectors allowing detection of FITC, TRITC, and cyanin-5 markers. Images were acquired sequentially for each fluorochrome to avoid fluorescence cross-talk among the 3 channels. The absence of cross-talk between channels captured in this way was confirmed by sections labeled with only 1 fluorophore. Sections (6 µm) from apo(a) transgenic mice and C57BL/6 mice on a high fat diet were triple-labeled with the following primary antibodies: Mac-1 for macrophages, smooth muscle -actin, and MCP-1 at the concentration as described above. TRITC-conjugated mouse anti-rat IgG (code 212-025-102; Jackson Immunoresearch) was used to detect macrophages by Mac-1 staining, cyanin-5 conjugated F(ab)₂ fragment rat anti-mouse IgG was used to detect smooth muscle- actin (code: 415-176-100; Jackson Immunoresearch), and FITC-conjugated donkey anti-goat IgG (code: 705-095-147; Jackson Immunoresearch) was used to stain for MCP-1. Each secondary antibody was affinipurified and was used at 30 µg/mL for TRITC and FITC and 36 µg/mL for cyanin-5 for 6 hours at room temperature.

Statistical Analysis
The significance of differences (probability value) between means was tested by use of the Mann-Whitney U test for nonparametric analysis. A probability value of 0.05 was taken to indicate statistical significance.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Macrophage Content of Lipid Lesions
First, we confirmed the previous observation that macrophages were absent from lesions in apo(a) transgenic mice²³ (Reckless J, Rubin EM, Verstuyft JB, Metcalfe JC, Grainger DJ. Unpublished data, 1998). Eight mice from each line were analyzed: 4 were fed a normal mouse diet, and the remainder were fed a diet with high fat content for 12 weeks from the age of 12 weeks onward. All mice were killed at 24 weeks of age. Macrophages were visualized with the use of 2 different rat monoclonal antibodies, Mac-1 and Mas034b. Consistent with previous reports^{23 39} (Reckless J, Rubin EM, Verstuyft JB, Metcalfe JC, Grainger DJ. Unpublished data, 1998), significant staining with both antibodies was seen in the aortic wall from C57BL/6 mice fed a high fat diet and apoE mice on either diet, with significantly greater staining in the apoE knockout mice (Table). The majority of the staining with both antibodies was colocalized with regions of lipid accumulation in C57BL/6 mice (data not shown), but macrophage staining was significantly more widespread in aortas from apoE knockout mice. In contrast, no staining was detectable with either antibody in the aortic wall from apo(a) transgenic mice, even when fed a high fat diet (Table). It should be noted that the amount of staining in each animal with the use of the 2 different anti-macrophage antibodies was very tightly correlated (r=0.972; P<0.0001), suggesting that the level of staining reflected the degree of macrophage infiltration rather than variations in the expression of either of the antigens by macrophages present.

View this table: [in this window] [in a new window]   Table 1. Quantitative Immunofluorescence Data for Each Mouse Line for Adhesion Molecules and Proinflammatory Cytokines

Thus, consistent with earlier studies with different anti-macrophage antibodies and electron microscopy,²³ we find that C57BL/6 mice fed a high fat diet have macrophage-rich lesions, whereas apo(a) transgenic mice fed a high fat diet develop lesions of similar severity but devoid of macrophages.

Adhesion Molecule Staining in Lipid Lesions
The levels of ICAM-1 and VCAM-1 in the vessel wall from mice of all 3 lines on both diets were determined by quantitative immunofluorescence.³⁷ Both ICAM-1 and VCAM-1 staining was significantly elevated in the vessel wall in all mice that developed lipid lesions (P<0.05 in all lines; apo(a) transgenic mice and C57BL/6 mice on a high fat diet and apoE knockout mice on either a high fat diet or normal diet). For all 3 lines, the severity of lipid lesion formation was exacerbated on a high fat diet, and there was a correlated increase in both vessel wall ICAM-1 and VCAM-1 staining in mice fed the high fat diet compared with mice fed the normal diet (Table and Figure 1). In contrast, there were undetectable levels of ICAM-1 staining in the aortic wall in mice that did not develop lipid lesions (C57BL/6 mice and apo(a) transgenic mice on a normal diet). Therefore ICAM-1 staining is only present in the vessel wall from mice in which lipid lesions have developed.

View larger version (52K): [in this window] [in a new window]   Figure 1. Distribution of adhesion proteins in the vessel wall of apo(a) transgenic and C57BL/6 mice. Consecutive 6-µm sections from a C57BL/6 (B, D, and F) and an apo(a) transgenic mouse (A, C, and E) fed a high fat diet for 12 weeks were stained for ICAM-1 and VCAM-1. A and B, Typical control sections (primary rat monoclonal antibody to ICAM-1 omitted). C and D, Representative ICAM-1 staining localized to the endothelial cell surface and in medial smooth muscle cells particularly close to the external elastic lamina. E and F, Representative VCAM-1 staining localized at the endothelial cell surface and in medial smooth muscle cells. ICAM-1 and VCAM-1 staining was elevated in lesions from apo(a) transgenic mice fed a high fat diet compared with C57BL/6 mice (Table).

ICAM-1 staining was localized in patches at the endothelial cell surface in apo(a) transgenic and C57BL/6 mice on the high fat diet (Figure 1), whereas in the more severely affected apoE knockout mice almost the entire endothelium was stained. Some cells in the media were strongly stained for ICAM-1, particularly in apo(a) transgenic mice on the high fat diet and apoE knockout mice on either diet. The pattern of VCAM-1 staining was very similar to that described for ICAM-1, although the number of positively stained medial cells was higher for VCAM-1 than ICAM-1 (Figure 1).

Proinflammatory Cytokine Staining in Lipid Lesions
We measured the levels of 3 representative proinflammatory cytokines, TNF-, MIP-1, and MCP-1, by using quantitative immunofluorescence³⁷ in neighboring sections to those stained for macrophages and adhesion molecules. As for the adhesion molecules, significant staining for TNF- was detected wherever lesions developed (apo(a) transgenic mice and C57BL/6 mice on a high fat diet and apoE knockout mice on either diet; Table and Figure 2). Furthermore, the amount of TNF- in the vessel wall was also higher in all mouse lines tested when fed a high fat diet compared with a normal diet (Table). Mice with no lesions (apo(a) transgenic mice and C57BL/6 mice fed a normal diet) had undetectable levels of TNF- staining in the vessel wall. TNF- staining was uniformly distributed throughout the vessel media in all mouse lines, and there was no localization either at the endothelial cell surface or at sites of lipid accumulation (Figure 2).

View larger version (43K): [in this window] [in a new window]   Figure 2. Distribution of proinflammatory cytokines in vessel wall of apo(a) transgenic and C57BL/6 mice. Consecutive 6-µm sections from a C57BL/6 (B, D, F, and H) and an apo(a) transgenic mouse (A, C, E, and G) fed a high fat diet for 12 weeks were stained for TNF-, MIP-1, and MCP-1. A and B, Typical control sections (primary goat polyclonal antibody to MCP-1 omitted). C and D, Representative TNF- staining is uniformly stained throughout vessel wall. E and F, Representative MIP-1 staining throughout vessel wall. G and H, Representative MCP-1 staining in each mouse line: C57BL/6 (H) shows uniform staining throughout vessel wall, whereas in apo(a) transgenic mice (G) MCP-1 is absent and staining is similar to control levels.

Staining for MIP-1 followed a pattern very similar to that for TNF-. MIP-1 staining was detectable only in mice that developed lesions (apo(a) transgenic mice and C57BL/6 mice on a high fat diet and apoE knockout mice on either diet). Similar to TNF-, MIP-1 staining was uniform throughout the vessel media in all mouse lines (Figure 2).

We also quantified the amount of staining for MCP-1 in the aortic vessel wall in all 3 mouse lines. Significant levels of MCP-1 staining were detected in the vessel wall from C57BL/6 mice on a high fat diet and apoE knockout mice on either diet (Table and Figure 2). However, in marked contrast to the cell adhesion molecules ICAM-1, VCAM-1, and the cytokines TNF- and MIP-1, no staining for MCP-1 could be detected in apo(a) transgenic mice (Table), even when fed a high fat diet (Figure 2G). We conclude that of the proinflammatory proteins we have studied, MCP-1 is selectively absent from the vessel wall of apo(a) transgenic mice during vascular lipid lesion development.

MCP-1 Staining Is Colocalized With Smooth Muscle Cells as Well as Macrophages
One possible explanation for the selective absence of MCP-1 in the macrophage-deficient apo(a) transgenic mice lesions is that the MCP-1 accumulation that normally accompanies lesion formation is synthesized exclusively by the macrophages. This explanation is unlikely because we have already shown that MCP-1 staining is uniform throughout the vessel media (for example, see Figure 2H), whereas in C57BL/6 mice macrophages are strongly localized to the focal lipid lesions. To further test this hypothesis, we used triple-label confocal microscopy to determine with which cell types in the vessel wall of C57BL/6 mice the MCP-1 was colocalized. Macrophages, identified by Mac-1 staining, were also stained strongly for MCP-1 (Figure 3). However, the MCP-1 staining was significantly more widespread than was the Mac-1 staining. In particular, smooth muscle cells (characterized by smooth muscle -actin staining; Figure 3) were also colocalized with MCP-1 staining (Figure 3). On the basis of 6 separate staining experiments followed by confocal microscopy, we estimate that more than half the cells stained positively for MCP-1 are smooth muscle cells (on the basis of smooth muscle -actin staining).

View larger version (38K): [in this window] [in a new window]   Figure 3. Colocalization of MCP-1 with macrophages and smooth muscle cells. Confocal microscopy was used to triple-label tissue sections from C57BL/6 and apo(a) transgenic mice fed a high fat diet as described in experimental procedures in text. Macrophages, identified by Mac-1 staining, were stained strongly for MCP-1 in C57BL/6 lipid lesions (A and B). In contrast, Mac-1 and MCP-1 staining was devoid of apo(a) transgenic mouse lipid lesions (D and E). However in C57BL/6 mice lipid lesions, MCP-1 staining was significantly more widespread than Mac-1 staining and smooth muscle cells (characterized by smooth muscle -actin staining, C) were also colocalized with MCP-1 staining (B). It is also interesting to note that as described previously,23 smooth muscle -actin staining is reduced in vessel wall of apo(a) transgenic mice compared with similar-sized C57BL/6 lipid lesions.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study we have exploited the observation that apo(a) transgenic mice develop vascular lipid lesions devoid of macrophages to identify which proinflammatory proteins might be involved in mediating monocyte recruitment during atherogenesis. Surprisingly, we observed that the macrophage-deficient apo(a) transgenic mouse lesions contained similar levels of several different proinflammatory proteins, both adhesion molecules and cytokines, to the macrophage-rich lesions of C57BL/6 mice. Consequently, we conclude that whereas ICAM-1, VCAM-1, TNF-, and MIP-1 may all be necessary for vascular monocyte recruitment in vivo, they cannot be sufficient. This is consistent with the observation of Patel and colleagues,⁴⁰ who demonstrated that monoclonal antibodies to VCAM-1 and ICAM-1 significantly reduced macrophage recruitment in apoE mice, whereas pretreatment with an E-selectin monoclonal antibody had no significant effect. Taking all these results together, we conclude that whereas VCAM-1 and ICAM-1 are necessary for vascular monocyte recruitment in vivo, they are not sufficient.

A number of recent studies have also demonstrated that TNF- alone was sufficient to induce local inflammation in a number of organs by injection of the recombinant protein. Similarly, antibodies to TNF- can abrogate inflammatory reactions in a number of experimental systems.^{41 42 43} Nevertheless, the levels of TNF- protein present in the lesions of apo(a) transgenic mice cannot be sufficient to induce vascular monocyte recruitment.

In marked contrast, MCP-1 protein was only detectable in the artery walls from mice that developed macrophage-rich lesions (C57BL/6 mice on a high fat diet and apoE knockout mice on either diet). MCP-1 protein was undetectable in the vessel wall taken from apo(a) transgenic mice on a high fat diet (Figures 2 and 3), even though lipid lesions of similar severity to those in C57BL/6 mice had developed. It is unclear the reason why MCP-1 production does not occur in the apo(a) transgenic mice during lipid lesion development. Previous studies of apoB/apo(a) double transgenic mice may shed some light on the mechanism: These double transgenics have lesions rich in macrophages⁴⁴ just like apoB overexpressing single transgenics, conclusively demonstrating that apo(a) has no dominant negative function on macrophage accumulation and therefore most likely not on MCP-1 secretion. Thus elevated expression of MCP-1 but not TNF-, MIP-1, ICAM-1, or VCAM-1 is correlated with vascular macrophage accumulation. We have not, however, determined whether MCP-1 is necessary for monocyte recruitment. It would be necessary to selectively inhibit MCP-1 function to demonstrate that MCP-1 was necessary for the inflammatory reaction that accompanies lesion development. This has been achieved for acute macrophage invasion models such as crescentic glomerulonephritis,⁴⁵ in which maximal macrophage recruitment occurs over a period of days with the use of anti–MCP-1 neutralizing antibodies.^{46 47} These experiments clearly show that monocyte recruitment in crescentic glomerulonephritis models is heavily dependent on MCP-1 activity.^{46 47} In contrast, monocyte recruitment during atherogenesis is a chronic process, even in the mouse models of the disease, occurring over a period of months.^{29 30 35 48} It is not presently feasible to inhibit MCP-1 activity for this period with the use of available antibodies. Similarly, the alternative approach of examining vascular monocyte recruitment in mice bearing a homozygous deletion of the JE/MCP-1 gene is also not presently feasible because the required deletion is embryonically lethal. To test the hypothesis that monocyte infiltration during atherogenesis is MCP-1 dependent, it will be necessary to develop specific pharmacological inhibitors of MCP-1 activity.

Our results suggest that chronic macrophage infiltration during vascular lipid lesion formation in mice may be dependent on MCP-1. Studies have shown that monocyte recruitment may depend on different proinflammatory cytokines, depending on the inflammatory stimulus provided. As noted above, monocyte recruitment in acute models of crescentic glomerulonephritis is largely MCP-1 dependent, whereas the monocyte recruitment that follows endotoxemia is not MCP-1 dependent but may result from TNF- or RANTES activity.^{41 42 49}

It is important to determine which of the many potentially proinflammatory proteins expressed during atherogenesis are necessary for monocyte recruitment in order to design agents that can inhibit the process in vivo. Our results highlight MCP-1 as a candidate protein for orchestrating vascular monocyte recruitment and hence suggest that pharmacological inhibitors of MCP-1 activity may be useful both as tools to probe the molecular mechanisms controlling inflammation in atherosclerosis as well as possible therapeutic agents capable of promoting atherosclerotic plaque stability.

	Acknowledgments

 
This work was funded by a program grant from the British Heart Foundation and a Wellcome Trust grant (to J.C.M. and D.J.G., who is a Royal Society University Research Fellow).

Received September 4, 1998; revision received December 7, 1998; accepted January 11, 1999.

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