The fate of monocytes in atherosclerosis

Circulating monocytes and their subsets

There is a growing recognition that subpopulations of monocytes may have differing roles in physiology and pathologies of diseases like atherosclerosis. Human monocytes are readily identified by the expression of CD14 [1,2]. In humans, CD14^hi monocytes comprise the major subset in the circulation, and CD14^int monocytes make up the typically far more infrequent subset. This less frequent subset is readily identified not only by expression of lower surface levels of CD14, but also by de novo expression of CD16 [1], the FcγRIII receptor, which plays a key role in recognizing immunocomplexes. These subsets also differentially express classical chemoattractant receptors like CCR2, the receptor for monocyte chemoattractant protein 1 [2].

Close counterparts to these populations have been studied particularly well in mice [2–7]. In mice, CD14 does not serve as a practical marker for identifying blood monocytes, as in humans. Instead, the expression of the macrophage colony-stimulating factor receptor CD115 (c-fms) selectively delineates monocytes in the blood of mice (this is also true for human). Staining for CD115 alone or in combination with F4/80 identifies the same subsets of monocytes in wild-type C57BL/6 mice as does the use of GFP knocked into the CX3CR1 locus [6], a popular model for tracing monocytes through an endogenous fluorescent tag [3,4,8]. In mice, staining for Ly-6C, a molecule of unknown functional significance at present, delineates monocyte subsets that resemble human monocyte subsets in patterns of chemokine receptor expression, like differential expression of CCR2, and at least some adhesion molecules [2]. Ly-6C^hi CD115⁺ monocytes in mice are considered to be the counterparts of human CD14^hi CD16⁻ monocytes, whereas Ly-6C^lo CD115⁺ mouse monocytes serve as counterparts to CD14^int CD16⁺ human monocytes. Recent Affymetrix gene expression analysis indicates that the analogy between species is vast, though not fully overlapping, and there is unrecognized conservation of molecules like the pattern of FcR expression between the human and mouse monocyte subsets that are deemed to be counterparts. Thus, it would be appropriate to refer to Ly-6C^lo mouse monocytes as ‘CD16⁺ monocytes’, the most popular term for the CD14^int monocytes in humans. A major difference between the mouse and human is the frequency of the CD16⁺ monocyte subset: this subset represents about half of circulating monocytes in mice [4], but <15% in healthy humans [1].

An argument has been developed that CD14^int CD16⁺ monocytes promote atherosclerosis, and there is consensus that this subset is associated with production of high levels of TNF in a variety of conditions in both humans [2] and mice [9]. If CD16⁺ human monocytes promote atherosclerosis, it remains unclear whether they do so by migrating into the plaque environment or by carrying out a role elsewhere. In mice, the two subsets are both recruited to atherosclerotic plaques, but CCR2^hi Ly-6C^hi monocytes, counterparts to the major subset in humans, are more robustly recruited to plaques [10,11]. The major CCR2^hi CD16⁻ human monocyte subset is equipped with higher surface expression of SR-A [12] and CD36 [13,14]. Despite this pattern of expression, uptake of oxidized LDL in humans is selectively mediated by the relatively infrequent subset of CD16⁺ monocytes [14]. It remains unclear if this activity affords CD16⁺ monocyte-derived cells an atheroprotective role wherein the cells dispose of oxidized LDL or a pro-atherosclerotic role. However, our recent unpublished findings suggest that Ly-6C^lo monocytes may potentially drive events associated with vulnerable plaque.

Recruitment into plaques and nontraditional roles of chemokine receptors

Three chemokine receptors – CCR2, CCR5, and CX3CR1 (also called fraktalkine receptor) – have received widespread attention as mediators of monocyte entry into atherosclerotic plaques [10,15,16]. In addition, new roles for other chemokines are continuously being identified [17]. Chemokine receptors may participate in atherosclerosis in ways that go beyond their role in attracting monocytes and other leukocytes into plaques. For example, chemokine-mediated signals control the frequency of monocyte within the circulation [7,18], and it may be the case that reducing overall monocyte frequency in the blood in turn affects plaque progression [16].

A nontraditional role for chemokine receptors is the regulation of cell survival. Recent studies have pointed strongly to a role for CX3CR1 in mediating survival of monocytes and monocyte-derived cells [19,20]. In particular, deficiency in CX3CR1 leads to a selective loss of Ly-6C^lo monocytes in the blood of mice [19,20]. In humans, it is known that the M280 polymorphism at the CX3CR1 locus is strongly atheroprotective [21–24]. Considering the discussion above regarding the differential role of monocyte subsets in atherosclerosis and the role of CX3CR1 in survival of Ly-6C^lo monocytes, there is a reasonable likelihood that the human M280 allele for CX3CR1 affects the survival of human CD16⁺ monocytes. It would be of great interest to determine if this is the case and to determine whether CD16⁺ monocyte frequency is elevated or reduced in M280 homozygotes. Clinical effort to investigate this question could greatly clarify views in the field about the potential role of CD16⁺ human monocytes in atherosclerosis. Overall, CX3CR1 antagonists may be attractive targets for atherosclerosis intervention [10], particularly since CX3CR1 is not widely required for inflammatory responses [8].

Migratory egress of foam cells from plaques

Another frontier in atherosclerosis research is the exploration of how foam cells may migrate out of plaques. In contrast to the chronic inflammatory status of atherosclerosis, acute inflammatory responses are often transient and a return to homeostasis is achieved within days to weeks [25–28]. The fate of monocyte-derived cells in inflammatory responses requires that they emigrate from the inflammatory site, enter lymphatics, and accumulate in draining lymph nodes [28]. The uptake of dying cells from tissues, including the large number of dying neutrophils found in acute inflammatory responses that resolve, may trigger migration of monocyte-derived cells to lymph nodes [29]. Some of the mediators released in this context that support emigration to lymph nodes are resolvin E1 and protectin D1 [29]. Reduced production of these mediators from atherosclerotic plaques may underlie a failed program of inflammatory resolution in atherosclerosis [30].

A few years ago, my colleagues and I demonstrated that migratory egress of monocyte-derived cells from plaques could be detected only under conditions of plaque regression [31]. Mobilization of monocyte-derived cells from plaques occurred through lymphatics in the adventitia and by return to the bloodstream, probably by migration of monocyte-derived cells directly across the arterial endothelium. Observations of mobilization out of plaques raised the possibility that restoring such mobilization, a normal feature of acute inflammatory responses, could lead to cholesterol transfer out of the artery wall and removal of cells that produce growth factors and cytokines that drive the disease forward. Furthermore, if mobilizing monocyte-derived cells out of plaques were associated with regression, then a new area for therapeutic development could arise in the quest for drugs that would promote this reponse.

The model used in these early studies was idealized for experimental investigation but not directly applicable to clinical scenarios; it involved surgical transfer of a plaque-bearing aortic segment from a mouse with atherosclerosis to another recipient mouse [31]. To study the issue of foam cell mobilization out of plaques, we have developed new techniques that will now allow for the testing of how various therapeutics may impact egress from plaques [32]. The principle of the new approach rests on tracking whether a nondegradable label brought into plaques by monocytes persists or disappears from plaques over time. The method first takes advantage of the high phagocytic/macropinocytic capacity of monocytes by labeling them with fluorescent latex beads intravenously [10]. The technique is akin to a pulse-chase experiment, in which the prolonged ‘pulse’ phase is comprised of the period of time in which monocytes carry the bulk of the label in the circulation and could be recruited to tissues, including but not limited to atherosclerotic plaques. Because latex-labeled monocytes undergo an expected turnover from the circulation, the ‘pulse phase’ in mice with atherosclerosis lasts for about 5 days [10]. Following this period, during the ‘chase phase,’ one can quantify the accumulation of label within plaques over time. If no phagocytes egress from plaques, then the quantity of label would persist indefinitely at the plateau reached after the pulse phase. However, if phagocytes leave plaques, some of the labeled phagocytes may be among them, and a loss of label would necessarily indicate egress from plaques, as the latex beads are not degraded and they could not be lost from plaques if the monocyte-derived cell that brought the label into the plaque died. Using this new technique, we have begun testing whether therapeutics like aggressive cholesterol lowering or administration of niacin or PPARγ agonists alters egress of phagocytes from plaques. These studies are still underway and will be discussed in the state-of-the art lecture. Only some therapies tested so far are able to induce emigration from plaques. One of the important benefits of inducing egress may be not only to modify plaque macrophage content but also to protect lesions from further growth of the necrotic core, a feature of vulnerable plaque.

Overall, it is clear that studies on the potential that ‘resolution programs’ can be restored in atherosclerosis is a frontier that holds promise for identifying new approaches to treatment, particularly for interventions that would affect established, advanced disease. Little is known about the mechanisms that would best facilitate the egress of foam cells from plaques, but the field is now positioned with the tools to make rapid progress in this area.