Labeling of blood monocyte subsets in apoE–/– mice.
Tracing the fate of monocytes in mice is challenging, as no tracking method is without drawbacks. Following adoptive transfer, only a very small fraction of transferred monocytes can be recovered (16
). Thus, large numbers of monocytes, pooled from many donors, are often transferred to overcome the relatively low sensitivity and quantitative nature of the method. Additional concerns include how isolation and manipulation of monocytes ex vivo affects subsequent differentiation and whether detection of a very small fraction of transferred cells accurately reflects behavior of the whole population (25
). Nonetheless, the approach has demonstrated utility, including in studies of monocyte migration to atherosclerotic plaques (26
). Knock-in of reporter genes to the CX3CR1 locus in mice (27
) has revealed monocyte subsets that endogenously express distinct GFP intensity in blood (15
). Monocytes can be tracked in these mice without adoptive transfer (15
), as we have previously done (19
); however, such tracking is limited to a short term after extravasation of monocytes, since subsequent differentiation can either reduce GFP expression to undetectable levels, such as in macrophages (27
), or elevate it, as in at least some DC populations (28
). Moreover, the knock-in mice inherently lack at least 1 allele of CX3CR1. As we set out to study the role of CX3CR1 in monocyte trafficking and to examine time points beyond 1–2 days, these problems precluded the use of this approach.
We have recently developed techniques to selectively label endogenous Ly-6Chi
monocytes i.v. through introduction of inert particulates (24
). These methods have the advantage of improved sensitivity and quantification of monocyte tracking over adoptive transfer, while yielding results that are in agreement with adoptive transfer approaches (29
). Particles that lack TLR ligands do not stimulate p38 MAPK activation or degradation of IκB in cultured macrophages (30
), so the extent of activation by inert latex beads in blood monocytes may be only minimal, but this is unclear. Although blood monocytes are predominantly labeled in the version of the method that labels Ly-6Chi
monocytes, a few neutrophils bear particles (24
), and other organs such as spleen and bone marrow do at least transiently harbor particulate-labeled cells that are not monocytes, including neutrophils and B cells (24
) (which are minimally or are not recruited to atherosclerotic plaques). Furthermore, labeling of Ly-6Chi
monocytes is conducted following prior depletion of monocytes via use of apoptosis-inducing liposomes (24
), and the full effects of the prior depletion are also not yet known. This drawback is similar to that in transgenically engineered mice where cell populations, such as DCs (31
) or macrophages (32
), are induced to undergo selective apoptosis through genetic manipulation. In the case of clodronate-loaded liposomes, apoptosis of the targeted cell population is restricted to the locale of liposome administration (33
), generally in contrast to transgenic models that lead to apoptosis systemically. However, so far, no major problems with the effects of prior DC or macrophage apoptosis on interpretation of subsequent experiments in these approaches have been described.
Given these potential drawbacks, before using the particulate labeling method, we carried out experiments in WT mice to determine whether the labeling methods affected known trafficking patterns of Ly-6Chi
monocytes or caused overt activation. We determined that engulfment of beads did not alter recruitment of monocytes to the acutely inflamed peritoneum, since accumulation of latex-labeled WT Ly-6Clo
monocytes in the thioglycollate-inflamed peritoneum (Figure A) mirrored the frequency predicted from studies that tracked unlabeled adoptively transferred Ly-6Chi
monocytes into the inflamed peritoneum (16
) (Figure B). We were able to distinguish resident macrophages from newly entering monocytes, because the latter express lower levels of F4/80 than resident macrophages (34
) (Figure A). This finding indicates that uptake of beads does not impair either subset of monocytes from emigrating out of the blood. In addition, migration of the latex+
subsets was low to absent in the noninflamed peritoneum, where the major population was resident F4/80hi
macrophages (Figure A), suggesting that the uptake of beads during labeling does not induce inflammatory patterns of extravasation.
Recruitment of labeled WT monocytes to peripheral sites of acute inflammation.
To assess whether administration of latex beads i.v. leads to substantial activation of monocytes, we stained monocytes for activation markers. CD62L is readily shed upon cellular activation (35
) and is expressed by Ly-6Chi
but not Ly-6Clo
). Staining for CD62L revealed that latex+
monocytes did not shed this activation-sensitive adhesion molecule (data not shown). Tissue factor is induced by many monocyte activation stimuli, but its expression and associated procoagulant activity were undetectable in the sera of latex-labeled mice (data not shown). Furthermore, we have carried out a whole mouse genome Affymetrix array (only 1 array has been completed in this ongoing analysis to date) by sorting latex–
monocytes from a pool of 5 female WT mice. The similarity of gene signatures, studied 24 hours after latex administration, between latex+
monocytes isolated from the same mice was greater (r2
= 0.9984) than the natural biological variability observed in another gene array comparison of sorted Ly-6Chi
monocytes from 2 separate sorts of unmanipulated mice (r2
= 0.9806). Thus, at least at the time point and condition examined to date, the carriage of latex bead(s) per se does not dramatically alter the monocyte.
We further reasoned that if overt activation is a major consequence of our labeling method, cytokine cascades associated with inflammation would be activated. Measuring cytokines in plasma would also allow us to detect whether any cells — not just monocytes — were activated enough to induce inflammatory gene expression. Thus, we measured the plasma levels of TNF-α, IL-6, and IFN-γ in naive animals (time 0, baseline) or 2, 12, and 24 hours after latex was introduced i.v. IL-6 was not detectable in any treatment (data not shown). Low levels of IFN-γ were found, but these were similar to baseline levels (Figure C). Although levels of TNF-α were mildly increased in the Ly-6Chi labeling protocol (Figure C), the difference was not statistically significant compared with baseline, and levels were near the detection limit of the ELISA. Thus, the particle labeling protocols did not trigger strong signs of monocyte or systemic activation, suggesting that monocytes or any other cells were not grossly activated by encounter with the beads. Taking all these data together, we conclude that the 2 techniques of selectively labeling Ly-6Chi and Ly-6Clo monocytes, respectively, could permit the design of experiments to trace the fate of monocyte subsets in atherosclerotic lesions.
To study the entry of monocytes from the circulation into atherosclerotic lesions, we labeled blood monocyte subsets in apoE–/–
mice. Intravenous injection of 0.5-μm latex particles efficiently and selectively labels monocytes in the circulation of WT mice (24
). In WT mice, 10%–15% of the monocytes are phagocytically labeled using this approach, and even after 5–7 days following initial labeling, 5%–7% of blood monocytes remain latex+
). We applied this technique to apoE–/–
mice. After i.v. injection of latex, 1%–2% of blood leukocytes acquired the fluorescent beads within 1 day, and 90% of these latex+
cells were CD115+
monocytes (Figure A). At day 1, 10% (range, 6%–13%) of blood monocytes were latex+
. The percentage of latex+
monocytes decreased over time, more rapidly than in WT mice (24
), to about 2%–2.5% (range 1%–3.5%) of blood monocytes being latex+
at days 3–7 (Figure A, and data not shown).
Labeling of monocyte subsets in apoE–/– mice.
mice, like WT mice (24
), i.v. injection of latex initially appeared in both monocyte subsets, but by 8 hours and thereafter, the vast majority of all circulating latex+
monocytes were Ly-6Clo
(Figure B) (24
). During the first several hours that followed i.v. administration of latex beads, we noticed that Ly-6Clo
monocytes in particular were often (Figure B), though not always (see Figure D in ref. 24
), transiently reduced. It is likely that these monocytes marginated in the vasculature, suggesting that they were at least transiently activated by the injection of beads. Thus, i.v. injection of latex particles labels a portion of Ly-6Clo
monocytes (approximately 10% of total monocytes, or 20% of the Ly-6Clo
subset) in apoE–/–
mice efficiently and specifically for more than 5 days (Figure C).
monocytes, on the other hand, can be stably labeled using a modification of this technique (Figure C), as previously described (24
), wherein monocytes are transiently depleted using clodronate-loaded liposomes prior to introduction of latex beads. The i.v. injection of clodronate-loaded liposomes initially fully depleted blood monocytes in apoE–/–
mice (Figure D; 4 hours after i.v. injection of latex beads i.v., 22 hours after i.v. injection of clodronate-loaded liposomes). Neutrophils and B cells that carry latex in the absence of monocytes donate the latex to bone marrow monocytes (24
), with a few neutrophils remaining latex+
in the circulation (ref. 24
and Figure ). Monocytes that returned to the circulation on day 2 were Ly-6Chi
, and 13% (range, 8%–17%) were latex+
(Figure D). Latex+
monocytes remained exclusively Ly-6Chi
for 4 days. At day 5, the percentage of latex+
monocytes had decreased by more than half, and apparent conversion of these monocytes to the Ly-6Clo
subset began to occur (18
) and was completed by day 7 (Figure , E and F). Early in the Ly-6Chi
labeling protocol, monocyte counts are reduced in the blood following depletion (24
), such that the normalized frequency of latex+
monocytes of each subset in the circulation indicates that latex+
monocytes were 3-fold more frequent in blood than Ly-6Chi
monocytes on the first day after labeling (Figure G) following the respective labeling strategies. Data were normalized by evaluating the fraction of total PBMCs that was latex+
at each time point, since total PBMC counts did not vary significantly in response to any of the experimental manipulations. By day 3 and thereafter, the normalized frequency of labeled monocytes was similar in the 2 subsets (Figure G).
Ly-6Chi, but not Ly-6Clo, monocytes employ CCR2 and CX3CR1 to enter atherosclerotic plaques.
We next studied the chemokine receptors used by monocyte subsets to enter and accumulate within atherosclerotic plaques. We expected Ly-6Chi
monocytes to use CCR2 to enter lesions. It is thought that Ly-6Clo
monocytes, which lack CCR2 (15
), utilize CX3CR1 for migration (16
), but Ly-6Chi
monocytes also express substantial levels of CX3CR1 (16
). Because both CCR2-deficient and CX3CR1-deficient mice have reduced plaque development when crossed with apoE-deficient mice (2
), we could not readily directly compare the rate of monocyte influx in these strains. Instead, we designed an experimental plan that permitted us to study the recruitment of WT, CCR2-deficient, and CX3CR1-deficient monocytes into atherosclerotic plaques that were derived uniformly from apoE-deficient CCR2+
mice. Specifically, we surgically transferred donor atherosclerotic aortic arches (36
) from apoE–/–
) into recipient mice that were CCR2+/+
, or CCR2+/+
and whose monocytes had been labeled with latex. Ly-6Chi
monocyte subset labeling efficiency did not differ in WT and KO mice (data not shown). Labeling of Ly-6Clo
monocytes was similar in CX3CR1–/–
and WT mice, but Ly-6Clo
monocytes showed reduced labeling in CX3CR1–/–
mice when carboxylate-modified beads were used, although this difference was not significant when plain polystyrene beads were used (as in the lesion analysis). However, CCR2–/–
mice had fewer circulating Ly-6Chi
monocytes, as reported previously (19
As expected, latex+ CCR2–/– Ly-6Chi monocytes were markedly less efficient than WT latex+Ly-6Chi monocytes at entering and accumulating within the grafted plaques (Figure A). The mean reduction in latex+ CCR2–/– Ly-6Chi monocytes in plaques was 72%, compared with WT controls (Figure A, left panel). However, when we accounted through normalization for the fact that Ly-6Chi monocytes exit the bone marrow less efficiently than WT monocytes, resulting in reduced numbers of circulating latex+ monocytes, there was only a 44% decrease in monocyte entry into plaques (Figure A, right panel), suggesting that CCR2 has 2 roles in mediating monocyte accumulation in atherosclerotic plaques: one effect is at the level of monocyte exit from the bone marrow; the other is emigration from blood into plaques.
Chemokine receptor utilization for entry of monocytes into atherosclerotic lesions.
monocytes entered plaques as well as WT monocytes (Figure B), which was expected, since this subset does not express CCR2 (16
). Unexpectedly, however, latex+
monocytes also did not require CX3CR1 to emigrate into plaques (Figure B), but latex+
monocyte entry into plaques was reduced by 54% in the absence of CX3CR1 (P
< 0.02) (Figure A). Thus, Ly-6Chi
blood monocytes utilize both CCR2 and CX3CR1 to enter atherosclerotic lesions. The CX3CR1 ligand CX3CL1 was detected on the endothelium overlying plaques (Figure C), consistent with the possibility that monocyte CX3CR1 may bind CX3CL1 on endothelium.
Role of CCR5 in monocyte migration to plaques.
To identify chemokine receptors that Ly-6Clo
monocytes use to enter plaques, we examined data from an ongoing preliminary gene array analysis that compared gene expression among monocyte subsets. CCR5 was selectively upregulated in apoE–/–
monocytes (data not shown), consistent with reports that the human CD16+
counterparts to these mouse monocytes preferentially express elevated levels of CCR5 (38
). To verify selective CCR5 upregulation in apoE-deficient Ly-6Clo
monocytes, we conducted real-time PCR for CCR5 and other chemokine receptors from sorted monocyte subpopulations. Indeed, CCR5 mRNA was selectively induced in Ly-6Clo
monocytes from apoE–/–
mice (Table ). Cell-surface expression of CCR5 was very weak relative to control staining in Ly-6Chi
monocytes, but expression was higher in Ly-6Clo
monocytes (Figure A). To determine whether CCR5 participated importantly in mediating entry or accumulation of monocyte subsets into plaques, we treated cohorts of apoE–/–
mice with anti-CCR5 neutralizing mAb (39
) or isotype control. Anti-CCR5 did not deplete circulating monocytes and did not significantly alter the frequency of Ly-6Chi
blood monocytes containing or lacking latex beads (data not shown). Nonetheless, there was a statistically significant inhibition of approximately 50% in Ly-6Clo
monocyte entry into plaques (Figure B). Thus, although Ly-6Clo
monocytes do not use CCR2 or CX3CR1 to enter atherosclerotic plaques, they are partially dependent upon CCR5 to do so. However, the antibody also partially blocked Ly-6Chi
monocyte entry into or early accumulation within plaques (Figure C), even though it is less obvious that Ly-6Chi
monocytes expressed CCR5. Thus, the effect of the anti-CCR5 neutralizing mAb was not limited to regulation of the trafficking of Ly-6Clo
Results of real-time PCR comparing chemokine receptor mRNA among monocyte subsets
Role of CCR5 in monocyte migration into plaques.