The secret life of nonclassical monocytes

Nomenclature of Monocytes

Current nomenclature divides monocytes into three subpopulations in humans and two subsets in mice. Human monocyte subpopulations are defined by their differential expression of CD14 and CD16, while mouse monocytes are classified according to differential expression of Ly6C. Classical monocytes (CD14⁺⁺CD16^– in humans, Ly6C^high in mouse) express high levels of CCR2 and migrate to sites of injury where they differentiate into inflammatory macrophages. Conversely, nonclassical monocytes (CD14⁺CD16⁺⁺ in humans, Ly6C^low in mouse) express high levels of the fractalkine adhesion receptor CX3CR1 and exhibit unique patrolling behavior along the resting vasculature 1 (see Fig. 1). A third subset of intermediate monocytes has also been described in humans (CD14⁺⁺ CD16⁺), which express multiple surface markers at levels between the classical and nonclassical subsets 2, and generally exhibit inflammatory functions. Previous studies grouped intermediate monocytes with the nonclassical subset due to their high expression of CX3CR1; however, increasing evidence suggests that intermediate monocytes are a third, distinct monocyte population in humans and do not patrol the vasculature 3. In mouse models of cardiac injury monocyte depletion reduces ventricular function and increases mortality 4, thus confirming that monocytes are critical for effective wound healing and repair of heart tissue following myocardial infarction (MI).

Figure 1

Monocyte subset dynamics and functions following myocardial infarction (MI). Following MI, monocytes released from the spleen and bone marrow migrate to the injured myocardium via the actions of chemokines. Classical monocytes (CD14⁺⁺CD16^– CCR2^high in humans, Ly6C^high in mouse) migrate to the site of infarct injury in response to a CCL2 chemokine gradient during the first phase of the monocyte response following MI (days 1–4). Classical monocytes differentiate into M1 proinflammatory macrophages, which exhibit phagocytic and proteolytic functions to remove debris in the injured infarct tissue. Nonclassical monocytes (CD14⁺CD16⁺⁺ in humans, Ly6C^low in mouse), express high levels of CX3CR1, and primarily exhibit patrolling behavior along the vasculature by responding to endothelial membrane-bound fractalkine and LFA-1. Nonclassical Ly6C^low monocytes are traditionally believed to infiltrate the myocardium in the second proliferative/reparative phase (days 5–9) of infarct healing although increasing evidence suggests an earlier role for this monocyte subset post-MI. Reported associations between monocyte subset counts in MI patients and clinical outcome suggest that this biphasic monocyte migration and differentiation may be harnessed to reduce the complications of myocardial ischemia such as adverse left ventricular remodeling.

Classical Versus Nonclassical Monocytes

It currently remains unclear whether nonclassical monocyte subsets arise from an independent bone marrow monocyte progenitor from that of classical monocytes or whether classical Ly6C^high CCR2^high monocytes serve as an intermediate lineage for the subsequent generation of nonclassical LY6C^low CX3CR1^high monocytes. Notably, human nonclassical CD14⁺CD16⁺⁺ monocytes also demonstrate patrolling behavior when infused in mice 5. The patrolling function of nonclassical monocytes is governed by the fractalkine/CX3CR1 axis and integrin adhesion molecules. Deletion of CX3CR1 reduces the patrolling of Ly6C^low monocytes, and results in overall fewer circulating nonclassical Ly6C^low monocytes 1, possibly due to reduced monocyte survival resulting from CX3CR1-dependent expression of antiapoptotic protein BCL-2. Alongside the fractalkine–CX3CR1 interaction, patrolling monocytes interact with the vascular endothelium via the lymphocyte function antigen-1 (LFA-1) comprising CD11a and CD18 integrins that interact with ICAM1 and ICAM2 on the surface of endothelial cells 1. Patrolling of nonclassical monocytes is a significantly slower process (∼12 µm/min) than the rolling behavior of classical monocytes and is independent of the direction of blood flow.

Clinical Implications of Monocyte Subset Counts in MI

Recent research has identified that human monocyte subset dynamics following MI hold predictive value for cardiovascular events and patient outcome post-MI. Broadly speaking, there are two types of MI, ST-elevated MI (STEMI) and non-ST elevated MI (NSTEMI), whose clinical presentation is comparable only regarding their onset, in that the unstable atherosclerotic plaque ruptures, so that platelets and erythrocytes are attracted and form a clot that can either block the complete blood supply (STEMI) or only partially constrict the artery and partly embolize into small areas of the left ventricle (NSTEMI). Percutaneous coronary interventional (PCI) therapy for STEMI needs to happen as fast as possible, as cardiomyocytes become necrotic 30–60 min after the onset of ischemia, whereas NSTEMI patients usually have a window of 72 h for PCI. Peak levels of classical CD14⁺⁺CD16^– monocytes post-MI were negatively associated with both the extent of myocardial salvage at 7 days post-MI and recovery of left ventricular ejection fraction during the chronic phase at 6 months post-MI 6. Another study of 100 STEMI patients demonstrated that monocytosis of intermediate CD14⁺⁺CD16⁺ monocytes during the acute phase after STEMI (days 1–7) predicted 2-year post-MI major adverse cardiac events following primary PCI 7. Other reports have identified that peripheral blood intermediate CD14⁺⁺CD16⁺ monocytes independently predict adverse cardiac outcomes of patients referred for elective coronary angiography 8. Such findings suggest that monocyte subpopulations specifically respond to the extent of cardiac injury and may offer reliable biomarkers to prioritize patients for follow up treatments/management. Importantly, it has not yet been investigated whether monocyte subpopulation responses in the very acute phase following MI (first 24 h) are predictive of cardiac outcome, since all research to date has focused on monocyte dynamics from day 1 onward following MI. Elucidating whether monocyte subsets have a pivotal role during the first 24 h following MI and their association with cardiac repair and patient outcome is of clinical importance since this may offer potential to therapeutically intervene while acute MI patients are still hospitalized.

The article by Leers and coworkers (DOI: cyto.a.23263; this issue, page 1059) investigated quantitative and qualitative changes in monocyte subsets in the first 6 h from onset of pain in over 80 patients with acute coronary syndrome (ACS) compared to 38 controls. While all monocytes were increased up to twofold across all subsets in ACS patients, phenotypic changes of the two most relevant markers (CD11b and CX3CR1) differed significantly in their expression between STEMI and NSTEMI patients (see Fig. 2). Due to their inherently different pathophysiology (inflammation in NSTEMI vs. ischemia, necrosis followed by inflammation in STEMI), one would also expect differences in the inflammatory response of monocytes. Leers and coworkers saw a clear increase in CD11b expression across all monocyte subsets in NSTEMI patients (inflammation), while CX3CR1 expression was significantly reduced in STEMI patients (ischemia). Independently, we also reported dramatic downregulation of CX3CR1 expression in monocytes as well as T lymphocytes in STEMI patients immediately after successful reperfusion through PCI, returning to baseline levels after 24 h 9. We identified two different mechanisms for this phenomenon in lymphocytes: transcriptional downregulation of CX3CR1 expression and reduced efficacy of the antibody used to detect the receptor in the FACS assay due to competition of soluble ligand (fractalkine, CX3CL1).

Figure 2

Monocyte subset dynamics and the expression of markers CX3CR1 and CD11b at 0–6 h following ST-elevated MI (STEMI) and non ST-elevated MI (NSTEMI). The clinical presentation of STEMI and NSTEMI is comparable only regarding their onset, in that the unstable atherosclerotic plaque ruptures, so that platelets and erythrocytes are attracted and form a clot that can either block the complete blood supply (STEMI) or only partially constrict the artery and partly embolize into small areas of the left ventricle (NSTEMI). While the dynamics and functions of monocyte subsets from day 1 following MI are well documented, very little is known about the role of monocytes during the very immediate acute phase during the first 24 h following MI. This study found an increase in all monocyte subsets in both STEMI and NSTEMI during the very acute phase at 0–6 h following MI, compared to patients without ACS or with an unstable angina pectoris (UAP). This study showed a clear increase in CD11b expression across all monocyte subsets in NSTEMI patients (inflammation), while CX3CR1 expression was significantly reduced in STEMI patients (ischemia). Such findings suggest that the myocardial ischemia generated by STEMI may lead to the downregulation of CX3CR1 during the very acute first few hours following MI. This knowledge of the change in expression of CX3CR1 and CD11b by distinct monocyte subsets between different ACS conditions and controls suggests that immunomodulation of the expression of such markers by monocyte subsets may hold potential as a therapeutic avenue for improving repair following MI.

Gating Strategy for Human Monocytes

Leers and coworkers (this issue) use a common gating strategy in their article by using CD14 versus CD16 scatterplots, followed by HLA-DR. While this backbone panel of markers allows the discrimination of human monocyte subsets, a more accurate gating strategy by polychromatic (multiparameter) FACS would include additional markers of other cells types in order to avoid confounders. Since NK cells and neutrophils also express high levels of CD16, CD56 (NK cells) and CD66b (neutrophils) should be employed to exclude such cell types, alongside CD3 and CD19 to exclude T cells and B cells, respectively. Such cell types must be excluded from analysis to ensure reproducible quantification. More recently, Shirk et al. demonstrate that the use of TLR2 in flow cytometry panels can improve the accuracy and quality of monocytes in circulating whole blood in both human and nonhuman primates, as TLR2 was found to be a stable expressed marker of all monocyte subsets during both steady state and immune activation 10. Furthermore, this study by Leers et al. used the rectangular gating (RG) strategy for the delineation of intermediate from nonclassical monocytes, which is reported by Zawada et al. to be a superior method over the trapezoid gating (TG) strategy 11. With regard to control measures, Leers and coworkers (this issue) included negative control samples, in which a portion of the cell suspension was incubated with directly conjugated (FITC, rPE, and PerCP-Cy5.5) nonrelevant isotype specific mouse immunoglobulins in addition to the panel of CD14, CD16, and CD45 backbone panel of antibodies. Such a control is important to allow for the correction of background staining by the conjugated primary antibody, as failing to block nonspecific binding can lead to erroneous results 12. This is particularly important when studying monocytes, since Fc receptors are expressed in highest abundance on monocytes/macrophages.

Conclusion

Classical monocytes are traditionally proinflammatory while nonclassical monocytes exhibit patrolling behavior along the vasculature via their high expression levels of the fractalkine receptor, CX3CR1. Nonclassical monocytes have also been reported to rapidly extravasate into tissues and are among the first responders to tissue injury releasing both TNFα and IL-1 to generate a proinflammatory milieu. Collectively, both murine and clinical studies indicate an intriguing role of classical, intermediate, and nonclassical monocyte subsets as predictive biomarkers of infarct healing. However, in order to understand this association of monocyte subset dynamics with MI outcome, a detailed understanding of the functions of different monocyte subsets at precise time points post-MI must be determined.