Chemokine Receptor CCR1 Disruption in Bone Marrow Cells Enhances Atherosclerotic Lesion Development and Inflammation in Mice
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Several chemokines or chemokine receptors are involved in atherogenesis. CCR1 is expressed by macrophages and lymphocytes, two major cell types involved in the progression of atherosclerosis, and binds to lesion-expressed ligands. We examined the direct role of the blood-borne chemokine receptor CCR1 in atherosclerosis by transplanting bone marrow cells from either CCR1+/+ or CCR1−/− mice into low-density lipoprotein-receptor (LDLr)-deficient mice. After exposure to an atherogenic diet for 8 weeks, no differences in fatty streak size or composition were detected between the 2 groups. After 12 weeks of atherogenic diet, however, an unexpected 70% increase in atherosclerotic lesion size in the thoracic aorta was detected in the CCR1−/− mice, accompanied by a 37% increase in the aortic sinus lesion area. CCR1−/− mice showed enhanced basal and concanavalin A-stimulated IFN-γ production by spleen T cells and enhanced plaque inflammation. In conclusion, blood-borne CCR1 alters the immuno-inflammatory response in atherosclerosis and prevents excessive plaque growth and inflammation.
Chemokines play key roles in the maturation, homing, and activation of leukocytes at sites of inflammation (1). Although various chemokines have been shown to be expressed in atherosclerotic arteries, so far only CCL2 (MCP-1) and CX3CL1 (Fractalkine) chemokines have been shown to play a direct role in atherosclerotic plaque development (2,3). Signaling through G protein-coupled chemokine receptors is responsible for chemokine effects on leukocyte development, recruitment, and activation (1). Three chemokine receptors, CCR2, CXCR2, and CX3CR1, appear to play key roles in leukocyte recruitment into the atherosclerotic vessels (4, 5, 6, 7). Many other chemokine receptors have been identified, however, and may play different roles in atherosclerosis (8). CCR1 is a chemokine receptor for CCL3 and CCL5 and is expressed on macrophages and T cells (8), two major cell types involved in atherosclerosis (9). In this study, we examined the direct role of blood-borne CCR1 in the development and composition of atherosclerotic plaques in low-density lipoprotein receptor-deficient (LDLr−/−) mice. We chose the irradiation/bone marrow transplantation model because CCR1 is mainly expressed on bone marrow-derived cells that give rise to the circulating leukocytes involved in atherogenesis (10).
Generation of Chimeric Mice
CCR1−/− mice on a C57BL/6J background were used (11). Female C57BL/6J LDLr−/− (Jackson Laboratory) were 7 months old. Mice were irradiated and reconstituted with CCR1+/+ or CCR1−/− (n = 10) bone marrow cells (5). After 4 weeks of recovery, mice were put on a diet containing 15% fat, 1.25% cholesterol, and 0% cholate for 8 weeks (n = 6 CCR1+/+; n = 7 CCR1−/−) to study fatty streak development or 12 weeks (n = 9 CCR1+/+; n = 10 CCR1−/−) to examine the development of large and advanced atherosclerotic plaques.
Assessment of the Extent of Atherosclerosis in Aortas and Aortic Sinus
Plasma total and HDL cholesterol levels were measured with a commercially available cholesterol kit (Sigma). Morphometric and immunohistochemical studies were performed in the aortic sinus and the thoracic aorta as previously described (12). Goat polyclonal antibodies against mouse CCL3 and CCR1 (Santa Cruz) were used at a dilution of 1:30 and 1:50, respectively, to assess CCL3 and CCR1 expression in atherosclerotic plaques. A goat anti-mouse IFN-γ antibody (R&D Systems) was used at 10 µg/mL, and a biotinylated EO6 antibody (provided by J. Witztum) that recognizes oxidized phospholipids was used at 8 µg/mL. Irrelevant immunoglobulins were used as negative controls. A semiquantitative score was established to compare IFN-γ staining between the two groups of mice. Maximal staining was scored as 3 and no staining as 0.
Murine cytokines (IL-2, IL-4, IFNγ, and TNFα) were measured in supernatants of mouse splenocytes and in plasma using the mouse Th1/Th2 cytokine cytometric bead array kit according to the manufacturer’s instructions (BD Biosciences).
Total RNA was extracted from splenocytes using the QIAamp RNA Blood Mini kit (Qiagen, Courtaboeuf, France) and cDNA generated with Superscript IITM Rnase H (Invitrogen, Carlsbad, CA, USA) following standard procedures. Amplification of the chemokine receptor transcripts was performed with kits for detecting the transcripts coding for the mouse CCR receptors CCR1, CCR2, CCR3, CCR4, and CCR5 and GAPDH (BioSource Europe) as previously described (13).
Data are expressed as means ± SE. Statistical significance was determined by ANOVA. A value of P < 0.05 was considered to be statistically significant.
The chemokines CCL3 and CCL5 are expressed in atherosclerotic lesions (16) and are thought to be major candidates for leukocyte recruitment into diseased arteries (8). CCL3 and CCL5 have two major receptors, CCR1 and CCR5 (1). A recent study has shown that CCR5 deficiency does not affect early atherosclerotic lesion development in apoE knockout mice (17). Thus, we hypothesized that deficiency in CCR1 expression may alter CCL3- and/or CCL5-induced monocyte recruitment and lead to reduction in lesion formation. Unexpectedly, we found that CCR1 deficiency in circulating leukocytes did not affect early fatty streak development, but rather promoted the progression toward larger and inflammatory atherosclerotic plaques at two different atherosclerosis-prone sites. These results suggest a novel and important role for CCR1 in the protection against excessive plaque growth.
Examining plaque composition, we found a significant and unexpected increase in the accumulation of macrophages within lesions of CCR1−/− mice after 12 weeks of atherogenic diet, suggesting that these cells were recruited through at least one of the other chemokine/chemokine receptor pathways previously reported to play a key role in leukocyte trafficking during this disease. At the advanced stages of lesion development, T lymphocyte accumulation within the lesion gradually decreases (14) as the plaque phenotype switches from an inflammatory toward a healing stage. However, despite the increase of plaque size in CCR1−/− mice, we found a persistent T cell accumulation within the lesions, testifying to the important immune activation in the absence of CCR1. This activated state was supported by our finding of enhanced conA-induced IFN-γ production in splenocytes of CCR1−/− mice and a lipid-associated increase in IFN-γ staining in spleen sections. Taken together, our results argue for an enhanced systemic Th1 immune response initiated by oxidized lipids that may have contributed to the exaggerated plaque inflammation and to the reduction in smooth muscle cell and collagen accumulation in advanced lesions of CCR1−/− mice.
Finally, total-body irradiation per se has been shown to differentially affect plaque size at two different atherosclerosis-prone sites (18). However, this is unlikely to affect our interpretation of the results, because both groups of mice received total-body irradiation. Moreover, differences in atherosclerotic lesion size were observed at the level of both the aortic sinus and the thoracic aorta.
In conclusion, we showed that rather than preventing leukocyte recruitment in atherosclerosis, the absence of CCR1 expression in leukocytes led to exaggerated IFN-γ production and promoted both lesion progression and inflammation, revealing novel roles for chemokine receptors in this complex disease.
This study was supported by the “Nouvelle Société Française d’Athérosclérose” (S.P.) and the “Fondation de France” (C.C.). We are grateful to Joseph Witztum and La Jolla SCOR program (Department of Medicine, University of California San Diego, La Jolla, CA, USA) for providing EO6 antibody.