Ly-6Chi monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata (original) (raw)
Hypercholesterolemic apoE–/– mice undergo gradual and systemic monocytosis of the Ly-6Chi subset. To test the hypothesis that high-fat feeding alters the repertoire of circulating monocytes, we analyzed peripheral blood mononuclear cells from C57BL/6 wild-type (referred to as apoE+/+) and apoE–/– mice that consumed either regular chow or Western diet (high in cholesterol and fat) for 25 weeks. Monocytes were defined as CD11bhiCD90loB220loCD49bloNK1.1loLy-6Glo mononuclear cells by flow cytometry, as previously reported (16), and further divided into Ly-6Chi and Ly-6Clo fractions (Figure 1A). apoE–/– mice on Western diet had a 4-fold increase of total circulating monocytes when compared with the same mice on chow (Figure 1B). Monocytosis in apoE–/– mice on Western diet resulted from a 14-fold increase of the Ly-6Chi subset (Figure 1C), whereas the Ly-6Clo population remained unchanged (Figure 1D). Consumption of a Western diet increased slightly the number of total circulating leukocytes in apoE–/– mice (mean ± SEM, chow, 3.0 ± 0.5 × 106 cells/ml; Western diet, 3.9 ± 0.4 × 106 cells/ml; Figure 1E). Blood smear counts showed that this increase arose primarily from monocytes (chow, 0.14 ± 0.03 × 106 cells/ml; Western diet, 0.94 ± 0.11 × 106 cells/ml), although granulocytes also increased (chow, 0.27 ± 0.03 × 106 cells/ml; Western diet, 0.96 ± 0.11 × 106 cells/ml) and lymphocytes decreased slightly (chow, 2.4 ± 0.6 × 106 cells/ml; Western diet, 2.0 ± 0.2 × 106 cells/ml). As expected (25, 26), apoE–/– mice on Western diet had increased serum cholesterol levels (479 ± 20 mg/dl) when compared with apoE–/– mice on chow (286 ± 25 mg/dl). Macroscopic and histologic examination of aortas revealed fatty streaks and fibrous plaque lesions in the root and descending aorta of apoE–/– mice regardless of diet. Lesions were identified along the entire aorta in older mice (i.e., mice that consumed Western diet for 50 weeks; data not shown). apoE–/– mice consuming Western diet had 3–5 times more extended and widespread atherosclerotic lesions than did apoE–/– mice on chow.
Hypercholesterolemia induces peripheral blood Ly-6Chi monocytosis. (A) Mononuclear cells from blood of apoE+/+ and apoE–/– mice consuming either chow or Western diet were stained with anti-CD11b, -CD90, -B220, -CD49b, -NK1.1, –Ly-6G, and –Ly-6C mAbs. Living cells were gated to determine presence and percentage of CD11bhiCD90loB220loCD49bloNK1.1loLy-6Glo monocytes (top row) and further divided into Ly-6Chi and Ly-6Clo subsets (bottom row). Representative dot plots and histograms from individual mice are depicted. Percentages of cells are shown as mean ± SEM. (B) Total blood monocytes in apoE+/+ and apoE–/– mice consuming either Western diet (+) or chow (–). (C) Total blood Ly-6Chi monocytes. (D) Total blood Ly-6Clo monocytes. (E) Total peripheral blood leukocytes. (F) Representative dot plots depicting expression of CD62L and CD11c among Ly-6Chi and Ly-6Clo monocytes. Percentages of cells in each quadrant are shown as mean ± SEM. (G) Representative cytospin preparations of purified blood Ly-6Chi and Ly-6Clo monocytes in apoE+/+ mice on chow and apoE–/– mice on Western diet. Scale bar: 10 μm. Student’s t test was used. Results are representative of 8 independent experiments with 5–14 mice per group.
apoE+/+ mice fed a Western diet had serum cholesterol levels of 227 ± 39 mg/dl, lower than those observed in apoE–/– mice on Western diet but higher than those in apoE+/+ mice on chow (101 ± 10 mg/dl). apoE+/+ mice fed a Western diet did not show a significant increase in the number of circulating monocytes or Ly-6Chi monocytes (Figure 1, B–D) and did not develop atherosclerotic lesions during the 25 weeks of diet consumption (data not shown).
apoE–/– mice also had elevated numbers of CD11b+CD90+B220+CD49b+NK1.1+Ly-6C+ cells (chow, 3.0% ± 0.1% cells; Western diet, 3.8% ± 0.4% cells) than apoE+/+ mice (chow, 1.8% ± 0.3% cells; Western diet, 1.9% ± 0.2% cells). These cells were phenotypically distinct from monocytes and were not examined further.
Although the number of circulating monocytes increased dramatically in apoE–/– mice fed Western diet, Ly-6Chi cells consistently expressed CD62 ligand (CD62L; also known as L-selectin) but not CD11c, while Ly-6Clo cells consistently expressed low levels of CD11c but not CD62L (Figure 1F), as previously reported for these monocyte subsets (21). Morphologic analysis of flow-sorted cells also showed that cells of both subsets retained their size as well as their characteristic kidney- or horseshoe-shaped nuclei (Figure 1G).
Having determined that numbers of circulating monocytes increased in apoE–/– mice on Western diet, we assessed the spatial and temporal course of monocytosis development by quantification of monocytes and their subsets in the bone marrow, peripheral blood, and spleen over 250 days of Western diet consumption (Figure 2A). Analysis included additional compartments because the bone marrow produces monocytes and the spleen may serve as a reservoir for monocytes in the periphery. Monocytosis developed progressively in all 3 compartments, and the blood and spleen showed predominant expansion of the Ly-6Chi subset. Statistical analysis matched the data to an exponential growth curve, permitting determination of doubling time for each tissue. The Ly-6Chi subset showed the lowest doubling times (95% confidence interval, 33 to 38 days in the blood), while, as expected, the Ly-6Clo monocyte subset had the highest doubling times (95% confidence interval, 145 to 256 days in the blood).
Peripheral blood monocytosis develops over the course of 250 days on an atherogenic diet. (A) Number of total monocytes and Ly-6Chi and Ly-6Clo subtypes in bone marrow, blood, and spleens of apoE–/– mice at various days of Western diet. Statistical analysis was based on an exponential growth curve and known cell numbers on day 0. Curve fit (solid line) and 95% confidence intervals (dashed lines) are shown. Doubling time (DT) of cell number is shown. (B) The same analysis was conducted with peripheral blood from apoE+/+ and apoE–/– mice that remained on chow diet. Doubling time of cell number in days is shown. (C) Splenic CD11bhiCD90loB220loCD49bloNK1.1loLy-6Glo cells were divided into F4/80hiCD11chiI-Ab–high macrophages/dendritic cells (gate i) and F4/80loCD11cloI-Ab–low monocytes, which were further divided into Ly-6Clo (gate ii) and Ly-6Chi (gate iii) subsets. These 3 subsets were isolated and stained with HEMA 3 for microscopic analysis. Scale bar: 10 μm. Results are pooled from 8 independent experiments.
Control experiments used blood from apoE+/+ and apoE–/– mice during 250 days of chow consumption (Figure 2B). apoE+/+ mice on chow did not develop monocytosis, excluding the possibility that age drives the increase. apoE–/– mice on chow showed moderate monocytosis. Statistical analysis matched the data to an exponential growth curve, though doubling times (e.g., 95% confidence interval, 63 to 86 days for Ly-6Chi monocytes in the blood) were 1.9–2.3 times longer than in apoE–/– mice on Western diet. Thus, atherosclerosis and Ly-6Chi monocytosis arise concomitantly in apoE–/– mice and show aggravation by Western diet.
Although we used the same criteria to define monocytes in the bone marrow and spleen as in blood (e.g., the CD11bhiCD90loB220loCD49bloNK1.1loLy-6Glo phenotype), the preponderance of macrophages and dendritic cells in the spleen necessitated a secondary step to ensure that the cells were indeed monocytes. In the spleen, monocytes were further defined as F4/80loCD11cloI-Ab–low. These cells had the same morphology as blood monocytes, whereas cells positive for F4/80, I-Ab, and/or CD11c resembled macrophages or dendritic cells (Figure 2C). Because of the low number of monocytes in the blood, the spleen therefore furnished a rich source of mononuclear cells for further study.
Monocytosis results from increased survival, continued proliferation and impaired Ly-6Chi to Ly-6Clo conversion. Given that the progressive and peripheral monocytosis of the Ly-6Chi subset in apoE–/– mice was most robust during consumption of Western diet, we next sought to determine whether a model modified lipoprotein, such as acetylated low-density lipoprotein (AcLDL), directly influences monocyte survival and/or proliferation. Initially, splenic Ly-6Chi and Ly-6Clo monocytes from apoE–/– mice on Western diet were isolated and cultured for 24 hours in medium supplemented or not with 100 μg/ml AcLDL. The presence of AcLDL allowed Ly-6Chi cells to survive (Figure 3A) while retaining their monocytic Ly-6ChiF4/80loCD11cloI-Ab–low phenotype. Conversely, survival of Ly-6Clo cells did not change in the presence of AcLDL (data not shown). Because LDL can induce aortic endothelial cells to synthesize and secrete M-CSF (27), we assessed whether this factor can also influence the fate of monocytes in vitro. Supplementation of medium with M-CSF (50 μg/ml) for 24 hours partially fostered Ly-6Chi monocyte survival (Figure 3A).
Ly-6Chi monocytosis results from increased survival, continued proliferation, and impaired Ly-6Chi to Ly-6Clo monocyte conversion. (A) Ly-6Chi monocytes from the spleens of apoE–/– mice were placed into culture with medium alone or medium supplemented with 100 μg/ml AcLDL or 50 μg/ml M-CSF. The percentage of cells alive 24 hours later was calculated based on the ratio of retrieved and input cell numbers. **P < 0.01, *P < 0.05 versus medium alone (1-way ANOVA with Tukey’s multiple comparison test). (B) apoE+/+ and apoE–/– mice on chow and Western diet received 3 i.p. injections of BrdU on 3 consecutive days. Cells from bone marrow, blood, and spleen were collected 1 day after the last injection and labeled with annexin V or anti-BrdU mAb. Results are shown for gated CD11bhiCD90loB220loCD49bloNK1.1loLy-6Glo monocytes as identified in Figure 1. Statistical analyses were performed using Student’s t test. (C) Representative dot plots depicting annexin V staining and BrdU incorporation in splenic CD11bhiCD90loB220loCD49bloNK1.1loLy-6Glo monocytes from apoE+/+ and apoE–/– mice on chow and Western diet. (D) apoE–/– mice on chow and Western diet received clodronate liposomes on day 0. Representative contour plots depict Ly-6C versus F4/80/I-Ab/CD11c phenotype among splenic CD11bhiCD90loB220loCD49bloNK1.1loLy-6Glo monocytes on days 1 and 5 or in age-matched untreated mice. (E) Percent of splenic Ly-6Clo monocytes recovered after clodronate liposome injection in apoE–/– mice on chow and Western diet compared with absolute number of cells in age-matched untreated mice. Shown are 1 of 2–3 independent experiments.
These data suggest that LDL and/or its derivatives directly and indirectly promote the survival of Ly-6Chi monocytes. Nevertheless, the in vitro conditions used may not reproduce the in vivo environment in apoE–/– mice on Western diet, since the number of Ly-6Chi monocytes did not increase in vitro. Therefore, we sought to compare directly the proliferation and survival of monocytes in vivo. apoE–/–animals consuming either chow or Western diet received daily i.p. injections of BrdU for 3 days and were sacrificed 1 day later. Mononuclear cells were purified from the bone marrow, blood, and spleen, and monocytes were analyzed for apoptosis (annexin V) and proliferation (anti-BrdU mAb). The Western diet decreased the number of annexin V+ cells within Ly-6Chi monocytes in the bone marrow and peripheral blood but not in the spleens of apoE–/– mice (Figure 3, B and C). The Western diet concurrently increased the number of BrdU+ cells within Ly-6Chi monocytes in peripheral blood and the spleen (Figure 3, B and C). The vast majority of bone marrow monocytes incorporated BrdU regardless of diet, likely reflecting the continuous medullary production of these cells. These observations reflect the increased survival of dividing Ly-6Chi monocytes in apoE–/– mice consuming Western diet, but may result from accelerated production in the bone marrow and/or from increased extramedullary proliferation. Interestingly, the higher levels of BrdU incorporation by Ly-6Chi monocytes in the spleen (mean fluorescence intensity, 528 ± 111) compared with the bone marrow (mean fluorescence intensity, 293 ± 63) indicate either continued proliferation in the periphery or selective emigration from the bone marrow of cells with longer proliferative histories.
Analysis of apoptosis and proliferation in apoE+/+ mice showed that Western diet increased survival and proliferation of monocytes in the spleen but not in the bone marrow or blood (Figure 3, B and C). Because apoE regulates apoptosis and cell cycle (28, 29), it may participate in the differences observed between apoE+/+ and apoE–/– mice.
To determine whether the Western diet also affects conversion of Ly-6Chi to Ly-6Clo monocytes, apoE–/– mice on chow and Western diet received clodronate liposomes to deplete endogenous circulating monocytes (24). Mice administered clodronate liposomes had a dramatically reduced number of Ly-6CloF4/80loCD11cloI-Ab–low monocytes in blood (chow, 0.6 ± 0.2 × 104 versus 8.7 ± 4.6 × 104 cells/ml; Western diet, 1.1 ± 0.6 × 104 versus 9.6 ± 1.3 × 104 cells/ml) and spleen (chow, 2.4 ± 1.1 × 104 versus 32.5 ± 7.3 × 104 cells/ml; Western diet, 3.8 ± 0.9 × 104 versus 381.0 ± 84.2 × 104 cells/ml) 1 day after injection. Clodronate also strongly reduced the number of Ly-6CloF4/80hiCD11chiI-Ab–high macrophages/dendritic cells and moderately reduced the number of Ly-6ChiF4/80loCD11cloI-Ab–low monocytes (Figure 3D and data not shown). The near absence of Ly-6Clo monocytes on day 1 allowed us to study their reemergence from the Ly-6Chi repertoire (Figure 3, D and E). Five days after clodronate injection, Ly-6Clo monocytes had repopulated both blood and spleen in animals on chow but not in animals consuming Western diet (Figure 3, D and E, and data not shown). Impaired Ly-6Chi to Ly-6Clo conversion in apoE–/– mice consuming Western diet fostered Ly-6Chi monocytosis.
Ly-6Chi monocytes accumulate selectively in atherosclerotic lesions. Adoptively transferred EGFP+ monocytes accumulate in atherosclerotic lesions (16), but low numbers of EGFP+ signals detected by immunohistochemistry prevent quantification of cell accumulation. Here we employed a recently established flow cytometry method (30) to phenotype single-cell suspensions of enzyme-digested aortas and determined the in vivo relevance of Ly-6Chi monocytosis to atherosclerosis. Aortas from apoE+/+ and apoE–/– animals on either chow or Western diet contained at least 2 distinct populations of cells expressing CD11b, identified in gate i as putative monocytes and in gate ii as putative macrophages (Figure 4, A and B). Cells detected in gate i fell into 4 phenotypically distinct populations: Ly-6ChiF4/80loCD11cloI-Ab–low, Ly-6ChiF4/80+CD11c+I-Ab–positive, Ly-6CintF4/80+CD11c+I-Ab–positive, and Ly-6CloF4/80loCD11cloI-Ab–low, resembling circulating Ly-6Chi monocytes, monocytes in the process of differentiation, differentiated macrophages and/or dendritic cells, and Ly-6Clo monocytes, respectively (Figure 4A).
Atherosclerotic lesions contain Ly-6Chi monocytes. (A) Aortas from apoE+/+ and apoE–/– mice on chow and Western diet were digested with a protease cocktail. Cells were dispersed and stained with anti-CD11b, -CD90, -B220, -CD49b, -NK1.1, –Ly-6G, -F4/80, –I-Ab, -CD11c, and –Ly-6C mAb. Percent (mean ± SEM) are shown for each quadrant. (B) Number of retrieved Ly-6Chi and Ly-6Clo monocytes per aorta in apoE+/+ and apoE–/– mice on chow and Western diet. Results are pooled from 5 independent experiments with 2–5 mice per group. Mean and SEM are shown. #P < 0.001 versus all other groups (1-way ANOVA with Tukey’s multiple comparison test). (C) Immunohistochemistry depicts the intima at the aortic root of a representative apoE–/– mouse on Western diet and an apoE+/+ mouse on chow. Sections stained with anti-CD31, –Ly-6C, and –Mac-3 mAbs are shown. Original magnification, ×400.
Enumeration showed 3,280 ± 240 Ly-6ChiF4/80loCD11cloI-Ab–low monocytes in the aortas of apoE–/– mice on Western diet, but only 580 ± 20 in the aortas of apoE–/– mice on chow. By comparison, aortas showed few Ly-6CloF4/80loCD11cloI-Ab–low monocytes (580 ± 70 and 500 ± 90 cells in apoE–/– mice on Western diet and chow, respectively; Figure 4B). Thus Western diet selectively increased Ly-6Chi monocyte accumulation in atherosclerotic aortas of apoE–/– mice. As expected, aortas contained more macrophages and/or dendritic cells in apoE–/– mice on Western diet (gate ii; 4.9 ± 1.7 × 104 cells) than on chow (1.1 ± 0.3 × 104 cells).
We counted relatively low numbers of Ly-6Chi and Ly-6Clo monocytes and macrophages/dendritic cells in the aortas of apoE+/+ mice regardless of diet (Western diet, 260 ± 10 Ly-6Chi monocytes, 310 ± 160 Ly-6Clo monocytes, 1.3 ± 0.5 × 104 macrophages/dendritic cells; chow, 220 ± 170 Ly-6Chi monocytes, 230 ± 120 Ly-6Clo monocytes, 1.2 ± 0.3 × 104 macrophages/dendritic cells; Figure 4B).
The above cell numbers likely underestimate actual values, since the enzymatic digestion of aortic tissue required to obtain single-cell suspensions caused the death of many cells (90% ± 1% of events contributed to debris and dead cells, as defined by low forward scatter) that were excluded from the analysis. The actual difference in Ly-6Chi and Ly-6Clo monocyte numbers is overestimated if Ly-6Clo monocytes are more likely to die during isolation.
We performed immunohistochemical examination of aortic roots isolated from the apoE–/– mice described above to evaluate the spatial distribution of Ly-6Chi cells in severe (fibrous plaque) and early (fatty streak) lesions (Figure 4C and data not shown). Ly-6C colocalized with mononuclear-like cells on the intimal face of CD31+ endothelial cells but not with the bulk of Mac-3+ macrophage-rich areas in both types of lesions. These observations suggest that Ly-6Chi monocytes migrate to early and severe lesions and that differentiation into macrophages accompanies transmigration into the artery, although some Ly-6Chi monocytes may reside in the innermost layer of the intima. The relative number of Ly-6Chi cells compared with the number of Mac-3+ cells in these regions was 4.8 times higher in severe than in early lesions, suggesting that Ly-6Chi monocytes migrate more efficiently to severe lesions.
Ly-6Chi monocytes adhere preferentially to activated endothelium, accumulate in atherosclerotic plaques, and rapidly become lesional macrophages. Further examination followed the activity and fate of Ly-6Chi and Ly-6Clo monocytes isolated from either apoE–/– or apoE+/+ mice on Western diet or chow. More than 70% of the cells remained alive in culture 24 hours after the isolation procedure (data not shown) and preserved monocytic markers (i.e., Ly-6Chi or Ly-6Clo, CD11bhiCD90loB220loCD49bloNK1.1loLy-6Glo, and F4/80loCD11cloI-Ab–low; Figure 5A). We initially determined the capacity of monocyte subsets to adhere to TNF-α–activated murine cardiac endothelium under laminar flow conditions. Freshly isolated blood Ly-6Chi monocytes from apoE–/– mice on either Western diet or chow adhered efficiently to the endothelium within minutes, while Ly-6Clo cells adhered significantly less well (Figure 5B, white bars), as did naive lymphocytes (data not shown). Similar results were observed with monocytes isolated from apoE+/+ mice (Figure 5B, black bars). These results indicate that Ly-6Chi monocytes adhere preferentially to activated endothelium independent of diet or of the presence or absence of apoE. The relative proportion of circulating Ly-6Chi and Ly-6Clo monocyte subsets in apoE–/– mice on Western diet suggests that greater than 95% of cells capable of binding to activated endothelium would belong to the Ly-6Chi subset (Figure 5C).
Ly-6Chi monocytes adhere preferentially to TNF-α–activated endothelium, accumulate in lesions, and differentiate to macrophages in vivo. (A) Purified Ly-6Chi monocyte phenotype after 24 hours in culture. (B) Adherence of blood monocytes on TNF-α–treated MHECs under laminar flow conditions. Monocyte subsets were isolated from the blood of apoE+/+ and apoE–/– mice on chow and Western diet. (C) Relative proportion of blood monocytes expected to adhere to activated endothelium, based on the capacity of each subset to adhere and their average abundance in peripheral blood of apoE–/– mice on Western diet for 25 weeks. (D) Ly-6C, F4/80, and I-Ab expression of CD45.2+ Ly-6Chi monocytes retrieved from aortas and spleens 24 hours after transfer into CD45.1+ mice (both donor and recipient apoE–/– mice consuming a Western diet). Monocytes cultured in vitro were also analyzed. (E) F4/80 and Ly-6C coexpression on CD45.2+ donor cells retrieved from aortas. (F) In vivo aortic accumulation of [111In]oxine-labeled Ly-6Chi and Ly-6Clo monocytes 24 hours after adoptive transfer in apoE–/– mice on Western diet. (G) Phosphorimager plates depicting relative distribution of signal in aortas of apoE–/– mice that received equal numbers of Ly-6Chi apoE–/– or Ly-6Clo apoE–/– monocytes. (H) Relative proportion of Ly-6Chi and Ly-6Clo monocytes expected to accumulate in atherosclerotic aortas, based on the capacity of each subset for aortic accumulation and their average abundance in peripheral blood of apoE–/– mice on Western diet for 25 weeks. Shown are 1 of 2–3 independent experiments. Student’s t test was used.
Further exploration used adoptive transfer of Ly-6Chi monocytes from CD45.2 apoE–/– mice into congenic CD45.1 apoE–/– recipients (both donor and recipient mice on Western diet for 25 weeks). Because peripheral blood contains few monocytes, splenic monocytes served as surrogates for circulating monocytes. After 24 hours, aortas from recipient mice were digested enzymatically, and single-cell suspensions were analyzed by flow cytometry. We counted 135 ± 4 donor cells in recipient aortas, of which approximately 25% were Ly-6Chi and approximately 75% were Ly-6Clo (Figure 5D). Many donor cells showed enhanced expression of F4/80 and I-Ab (Figure 5D). Remarkably, the combined analysis of Ly-6C and F4/80 expression by donor cells revealed the existence of at least 3 distinct populations, Ly-6ChiF4/80–, Ly-6ChiF4/80+, and Ly-6CloF4/80+, resembling monocytes, monocytes in the process of differentiating into macrophages, and mature macrophages, respectively (Figure 5E). These phenotypic relationships are comparable to those observed among endogenous populations (Figure 4A). In contrast, donor cells retrieved from the spleens of the same mice remained phenotypically unchanged (Figure 5D). These results demonstrate recruitment of Ly-6Chi monocytes to atherosclerotic aortas, followed by local and rapid (<24 hours) differentiation into macrophages. Combined with the restricted localization of Ly-6Chi monocytes to the luminal face of the endothelium, these data suggest that differentiation into macrophages accompanies transmigration and also support the notion that aortic monocyte detection did not result from contamination of circulating cells.
To determine the relative capacity of Ly-6Chi and Ly-6Clo monocytes to migrate to atherosclerotic aortas and to determine whether accumulation of Ly-6Chi cells mapped to lesions, we labeled equal numbers of splenic Ly-6Chi and Ly-6Clo monocytes from apoE–/– mice on Western diet with [111In]oxine and injected them separately into apoE–/– mice on Western diet. After 24 hours, we excised the aortas and calculated the percent injected dose per gram of tissue, which revealed that Ly-6Chi cells preferentially accumulated in aortas (Figure 5F). Autoradiography showed discrete regions of activity only in recipients of Ly-6Chi cells (Figure 5G). The lack of such regions in animals receiving Ly-6Clo cells suggests that the signals detected in these mice corresponded to background activity. The radioactive signal observed for Ly-6Chi cells always mapped directly to areas containing lesions as determined microscopically, but not all lesions showed focal areas of radioactivity (data not shown). The relative proportion of circulating Ly-6Chi and Ly-6Clo monocyte subsets in apoE–/– mice on Western diet suggests that greater than 90% of cells accumulating in atherosclerotic lesions originate from the Ly-6Chi subset (Figure 5H). Taken together, these results suggest that circulating Ly-6Chi monocytes are direct precursors of lesional macrophages.
Expression of MCP-1 by a subset of cells in atherosclerotic lesions suggests active recruitment of monocytes to developing lesions in vivo (31, 32), and mice lacking MCP-1 or C-C motif chemokine receptor 2 (CCR2) show reduced atherosclerosis (33–35). Thus monocyte recruitment into lesions may require CCR2 expression, a feature of the Ly-6Chi subtype (19). Testing this hypothesis involved the adoptive transfer of Ly-6Chi monocytes from bone marrow of CCR2–/– mice into peripheral blood of atherosclerotic (CCR2+/+) apoE–/– mice. We counted only 14 ± 2 donor cells in recipient aortas, suggesting that Ly-6Chi monocyte accumulation in lesions does indeed depend on CCR2.
Statin administration attenuates Ly-6Chi monocytosis. Having shown that lesional macrophages were derived from circulating Ly-6Chi monocytes in atherosclerosis, we sought to determine whether reduction of Ly-6Chi monocytosis attenuates disease. Repeated administration of anti–Ly-6C mAb could theoretically control Ly-6Chi monocytosis. This approach is impractical, given the chronic nature of atherogenesis, and may not keep the size of the Ly-6Chi monocyte population at homeostatic levels, but rather trigger transient cell depletion upon each mAb injection. In contrast, since inhibitors of hydroxymethylglutaryl coenzyme A reductase (statins) decrease cholesterol levels, exert antiinflammatory effects, and attenuate atherosclerosis (36, 37), we sought to determine whether concurrent treatment of apoE–/– mice on Western diet with statin also modulates the extent of monocytosis. Mice analyzed after 25 weeks of atorvastatin treatment had significantly attenuated serum cholesterol levels when compared with age-matched littermates on Western diet (Figure 6A). The statin treatment also reduced monocytosis (Figure 6B). Specifically, the numbers of Ly-6Chi monocytes declined significantly in the spleen and peripheral blood (although they remained higher than in apoE–/– mice on chow), whereas the numbers of Ly-6Chi monocytes in the bone marrow fell to levels found in apoE–/– mice on chow (Figure 6B). The statin treatment also reduced the numbers of the Ly-6Clo monocytes in the spleen. Antiinflammatory effects of statins beyond LDL lowering could also participate in the attenuation of Ly-6Chi cell numbers.
Statin treatment limits monocytosis. apoE–/– mice consumed Western diet supplemented or not with atorvastatin for 25 weeks. A control group of apoE–/– mice received regular chow. (A) Serum cholesterol after 25 weeks of diet. (B) Number of leukocytes, monocytes, and Ly-6Chi and Ly-6Clo subtypes in bone marrow, blood, and spleen. (C) Association between serum cholesterol and number of circulating Ly-6Chi or Ly-6Clo monocytes after 25 weeks of diet. Mean ± SEM are shown for apoE–/– mice on chow (filled circles), Western diet (open circles), and Western diet supplemented with atorvastatin (gray circles). Results are pooled from 9 independent experiments (n = 3–14 per group). Student’s t test was used.
Blood Ly-6Chi monocyte counts positively associated with serum cholesterol levels in apoE+/+ (data not shown) and apoE–/– mice (Figure 6C). Furthermore, correlation between Ly-6Chi numbers from statin-treated apoE–/– mice conformed to a linear axis between chow- and Western diet–fed animals. Such correlative analysis did not apply for blood Ly-6Clo monocytes, since Ly-6Clo cell counts were similar for all serum cholesterol concentrations.