In this study, we sought to determine whether CX3CL1 and CCR2 act independently or in concert to promote lesion formation through their effects on macrophage accumulation. There are 2 major findings in the present study. First, although deletion of either CX3CL1 or CCR2 reduced macrophage presence and atherosclerosis, atheroprotection in the CCR2−/− mice was more robust. Second, there was an even greater reduction of lesion formation in the CX3CL1−/−CCR2−/− double knockouts, which suggests that CX3CL1 and CCR2 have independent functions in recruiting monocyte/macrophages. These differences, which were not linked to changes in cholesterol levels or lipoprotein profiles, provide mechanistic insights into the role of monocyte chemoattractants in fatty streak formation and early plaque development.
The past 2 decades have witnessed a rapid increase in our understanding of leukocyte migration throughout the body, with the identification of ≈50 chemokines and >20 chemokine receptors. Several chemokines and their cognate receptors have been implicated in atherogenesis, including KC/CXCR2,15
The finding that deletion of individual ligands or their receptors reduces lesion size suggests that these chemokines have specific roles in recruiting and retaining monocytes in the vessel wall; however, the sum of the reductions reported for the individual knockouts easily exceeds 100%, and so, they cannot all be acting independently.
Several lines of evidence support the notion that CCL2/CCR2 and CX3CL1/CX3CR1 are the primary chemokine/receptor pairs that drive atherosclerotic lesion formation. A single-nucleotide polymorphism (SNP) in CCL2 that results in increased plasma levels has been associated with increased risk of myocardial infarction,21,22
as has a CCR2 SNP (V64I).23
Two SNPs (I249V and T280M) have been identified for CX3CR1 and linked to protection from coronary artery disease.24,25
In the mouse, genetic deletion of either CCL218,19
reduced diet-induced lesion formation by at least 50%. Similarly, deletion of CX3CR1 robustly reduced lesion size and macrophage recruitment in the aorta.11,12
We sought to determine whether these 2 chemokine/receptor pairs were acting independently or in concert in promoting lesion formation. CCR2 and CX3CR1 are closely linked on the same chromosome, and it is difficult to create double-receptor knockouts. We took advantage of the fact that CX3CL1 is the only known ligand for CX3CR1 and crossed CX3CL1−/− and CCR2−/− mice to investigate the combined effect of the loss of both of these signaling pathways. After 8 weeks on the Western diet, the mice were examined for populations of circulating blood monocytes, recruitment of monocyte/macrophages to the artery wall, and atherosclerotic lesion size in each of the 4 groups.
Recent work identified a population of circulating monocytes that are recruited from the blood to developing atherosclerotic lesions.9,27
By fluorescence-activated cell sorter analysis, these monocytes are Ly6Chi
and express CCR2.6,7
mice, this population is greatly reduced in size due to a failure of monocytes to leave the bone marrow, particularly in the setting of infection6
and their scarcity in the blood may contribute to the impaired macrophage recruitment and atheroprotection seen in CCR2−/−
mice. Before the present study, few data were available on monocyte populations in CX3CL1−/−
mice, and none were available in the setting of hypercholesterolemia. In agreement with earlier work, we found a marked decrease in the number of Gr1hi
monocytes in the CCR2−/−
mice fed the high-fat diet.7
In contrast, the Gr1hi monocytes were normal in CX3CL1−/−
mice, and significantly, deletion of CX3CL1 did not further decrease this population in CCR2−/−
mice. Thus, any differences seen in macrophage accumulation between CCR2−/−
mice cannot be ascribed to differences in monocyte numbers.
Macrophage content in atherosclerotic lesions was similar in WT and CX3CL1−/−
mice, although there was a trend toward a reduction in the CX3CL1−/−
mice. As expected, markedly fewer macrophages were found in the lesions in CCR2−/−
mice. Tacke et al9
showed that monocytes require CCR2 and CX3CR1 (the receptor for CX3CL1) for direct recruitment to lesions, but it was unclear whether the receptors functioned in sequence or independently. Significantly, we show that deletion of CX3CL1 further reduced macrophage presence in the lesions of CCR2−/−
mice. This additive phenotype provides in vivo evidence for independent contributions of CX3CL1 and CCR2 in monocyte/macrophage recruitment to the artery wall. Because this phenotype cannot be attributed to a reduction in circulating monocyte levels, it suggests that CX3CL1 is acting locally at the site of recruitment.
We next asked whether the decreased macrophage recruitment in CX3CL1−/−
double knockouts led to a reduction in atherosclerotic lesion size. As part of this study, we directly compared the extent of atherosclerosis in CX3CL1−/−
mice, both by en face
staining of the entire aorta and by staining serial sections through the aortic root. By both methods, the atheroprotection afforded by deletion of CCR2 was significantly greater than deletion of CX3CL1. Similarly, Teupser et al20
found only modest reductions in the aortic root of CX3CL1−/−
mice. Nonetheless, deletion of CX3CL1 in the CCR2−/−
mice resulted in a further reduction in lesion size in both the total aorta and in the aortic root, in both males and females, which indicates an additive or synergistic effect. These results are consistent with the macrophage staining results and suggest that CX3CL1 and CCR2 act independently to recruit monocyte/macrophages to the vessel wall at an early step in the development of atherosclerotic lesions. Differences in cholesterol levels or weights of the mice cannot account for these results. In fact, both the weights and total plasma cholesterol levels were greater in the knockout mice than in the WT mice. For example, the CX3CL1−/−
mice were heavier and had slightly higher plasma cholesterol levels than the CCR2−/−
mice, yet they had by far the least atherosclerotic lesion formation.
At least 2 distinct models can be envisioned to explain chemokine-dependent recruitment of monocytes to atherosclerotic lesions. In the first, a chemokine such as CCL2 functions as a chemoattractant to bring monocytes to the vessel wall, where they are then localized and captured by other chemokines, such as CX3CL1 and interleukin-8, which are competent cell-adhesion molecules. In this multistep sequential model, different chemokines have distinct but interdependent functions, and genetic deletion of any 1 in the sequence would be expected to produce a similar phenotype (ie, deletion of both chemokines would be no more protective than the individual deletions). Barlic et al28 suggested such a mechanism, based in part on the in vitro findings that oxidized lipids downregulate expression of CCR2 on monocytes but upregulate CX3CR1 expression. However, in vivo data in the present study demonstrate that the simultaneous loss of CCR2 and CX3CL1 results in additive reductions in macrophage recruitment and atheroprotection and thus argue against such a sequential, interdependent recruitment mechanism.
In a second model, CX3CL1 and CCL2 act independently to recruit either the same or different monocytes to the artery wall. Geissmann et al8
described 2 populations of blood monocytes: 1 population is Gr1+
, and the other is Gr1−
. Tacke et al9
found that both CCR2 and CX3CR1 contributed to migration of the Ly6Chi
monocytes to atherosclerotic lesions, and surprisingly, CCR5 (but not CX3CR1) was required for accumulation of the Ly6Clo
Because the deletion of both receptors markedly reduces macrophage accumulation in lesions, the present data suggest that CCR2 and CX3CR1 are recruiting different monocytes within the Ly6Chi
population. CCL2 and CX3CL1 are expressed in both endothelial cells and smooth muscle, but CX3CL1 is particularly abundant in medial smooth muscle cells.11,28
Different monocytes may thus be recruited to different locations, and the location might depend on where they first encounter chemokines. This mechanism is consistent with a model in which CCR2 and CX3CR1 act independently and nonsequentially.
These results have several clinical implications. First, with regard to atheroprotection, CCR2 appears to be a more promising therapeutic target than CX3CL1. Second, for therapeutic purposes, it might be necessary to block both CX3CL1 and CCR2. In this regard, Martin et al29
recently reported that the viral-encoded protein M3, which binds a broad range of chemokines, provided excellent protection against development of streptozotocin-induced diabetes in mice. Redundancy has been an issue in the chemokine family since the full spectrum of the ligands and receptors became apparent. The present study suggests that it might be necessary to simultaneously block 2 or more chemokine receptors to achieve robust clinical results.
The past decade has witnessed a dramatic increase in our understanding of the importance of inflammation in all stages of atherosclerotic heart disease. Subendothelial foam cells, the hallmark of early lesions, are derived from circulating blood monocytes, and recent evidence indicates that chemokines play important roles in directing monocyte migration from the blood to the vessel wall. Genetic deletions of monocyte chemoattractant protein-1 (MCP-1, CCL2), fractalkine (CX3CL1), or their cognate receptors, CCR2 and CX3CR1, have been shown to markedly reduce monocyte recruitment and atherosclerotic lesion size in murine models of atherosclerosis; however, whether these 2 chemokine systems were redundant or made independent contributions was unknown. To address this question, we created double knockouts of fractalkine and CCR2 (on the apolipoprotein E–null background) and performed a 4-arm atherosclerosis study. The results demonstrate that deletion of CCR2 affords significantly more protection than deletion of fractalkine. Significantly, deletion of fractalkine in CCR2-null mice further reduced monocyte recruitment and atherosclerotic lesion size, which indicates that both chemokines make independent and significant contributions to atherogenesis. These data provide the first in vivo evidence for independent roles for CCR2 and CX3CL1 in monocyte recruitment and atherosclerotic lesion formation and suggest that successful therapeutic strategies for atherosclerosis or other classic inflammatory diseases such as rheumatoid arthritis or multiple sclerosis may need to target multiple chemokines or chemokine receptors.