What is a monocyte?

Monocytes were first described in 1882 by Ilya Metchnjkoff and have since been demonstrated to be critical in human health and disease [1]. Monocytes are a leukocyte subset that play key roles in homeostasis, pathogen challenge and clearance, and inflammation. Monocytes represent approximately 4% of total leukocytes in mice and 10% in humans [2,3]. After production and development from progenitors in the bone marrow, monocytes circulate in the vasculature, bone marrow, and spleen; they do not proliferate in a steady state. Whether in the normal course of development or due to pathogen challenge, once monocytes migrate to and into peripheral tissues, they differentiate into dendritic cells or macrophages. Plasticity and heterogeneity are monocyte hallmarks, as they can rapidly adjust their functional phenotype in response to changing immunological cues.


Monocyte development

Monocytes are members of the mononuclear phagocyte system (MPS), which is a comprehensive classification of all highly phagocytic mononuclear cells and their precursors [3,4,5]. The MPS comprises all myeloid immune cells other than polymorphonuclear granulocytes. The origin and lineage of the cells in the MPS remained poorly understood until the availability of multicolor fluorescence-activated cell sorting (FACS), which allows identification of progenitors and differentiated cell populations based on the expression of specific cellular markers [4,5]. Current models of monocyte differentiation propose that circulating monocytes originate from hematopoietic stem cell (HSC)-derived progenitors with myeloid restricted potential [4,5]. Subsequent commitment steps during monocyte differentiation in the bone marrow involve common myeloid progenitors (CMPs), granulocyte-macrophage precursors (GMPs), and macrophage and DC precursors (MDPs) (Figure 1) [5,6].

Monocyte subset markers

Human monocyte markers

Expression of CD14, CD16 (Fcγ RIII), CD64 (Fcγ RI), and the chemokine receptors CD192 and CX3CR1 define human peripheral blood monocyte populations into three different subsets: classical, intermediate, and nonclassical (Table 1). These subpopulations can be further characterized by different levels of HLA-DR and CD195, as well as of the receptors TNFR1 (CD120a) and TNFR2 (CD120b). TNFR1 expression is highest on intermediate monocytes, whereas TNFR2 expression is highest on nonclassical monocytes [5,6,7,8].

Table 1. Markers for human monocytes.

Population Marker Chemokine receptors Function
Classical
87–88%
CD14Hi
CD64
CD62L
TNFR1
TNFR2Low
CD192Hi
CXCR1Low
Primary population for phagocytic activity
Low pro-inflammatory cytokine production
Intermediate (classical CD16+)
2–3%
CD16
CD14Hi
CD64
HLA-DRHi
TNFR1Hi
TNFR2
CD192Low
CX3CR1Hi
CD195
Pro-inflammatory
Produces TNF-a, IL1b, and IL-6
Nonclassical
10%
CD14Low
CD16Hi
TNFR1Low
TNFR2Hi
  Anti-inflammatory
Constitutively produces IL-1RA
Abbreviations: CD, cluster of differentiation; CXC, cysteine–any amino acid–cysteine; CXCR, CXC chemokine receptor; HLA, human leukocyte antigen; IL, interleukin; TNF, tumor necrosis factor. TNFR, tumor necrosis factor receptor. Low: low expression levels, Hi: high expression levels


Mouse monocyte markers

Mouse monocytes also have three distinct subpopulations (Table 2), defined by their cell surface expression of Ly6C, CD11b, and chemokine receptors CD192 and CX3CR1. In addition, a high level of CD115 expression discriminates between peripheral blood monocytes and granulocytes, as well as lymphocytes, which also express CD11b (Mac-1) [5,6,8].

Table 2. Markers for mouse monocytes.

Population Marker Chemokine receptors Function
Classical
Ly6CBright
40%
Ly6CHi
CD43Low
CD11b
CD115
CD62L
CD192Hi
CXCR1Low
Inflammation produces TNFa
Intermediate
20%
Ly6CHi
CD43Hi
CD11b
CD115
  Pro-inflammatory
Nonclassical
Ly6CDim
Ly6CLow
CD43Hi
CD11b
CD115
CD11c
CD192Low
CX3CR1Hi
Anti-inflammatory
Constitutively produces IL-1RA
Abbreviations: CD, cluster of differentiation; CXC, cysteine–any amino acid–cysteine; CXCR, CXC chemokine receptor; HLA, human leukocyte antigen; IL, interleukin; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor. Low: low expression levels, Hi: high expression levels


Monocyte trafficking

Chemokines and monocyte trafficking

CCR2

CCL2 (also known as MCP1) and CCL7 (also known as MCP3) are CC chemokines that bind to CCR2 and mediate Ly6CBright monocyte recruitment [9,10,12]. Although expression of CCR2 is restricted to only a few cell types, most (if not all) nucleated cells express CCL2 in response to activation by pro-inflammatory cytokines or stimulation of innate immune receptors by a range of microbial molecules. Many infections induce the expression of CCL2 and result in high circulating levels of CCL2 in serum and within inflamed tissues. CCL2 dimerizes and associates with tissue glycosaminoglycans, establishing gradients that guide monocytes towards sites of infection or inflammation. In support of this model, monocyte recruitment is impaired by amino acid substitutions in CCL2 that prevent dimerization or association with glycosaminoglycans.

CCR1 and CCR5

Monocytes also express CCR1 and CCR5, and these receptors bind to a variety of chemokines, including the shared ligands CCL3 (also known as MIP1α) and CCL5 [11,12]. In vitro transmigration assays have shown: (1) CCR1 predominantly mediates the arrest of monocytes in the presence of shear flow; (2) CCR5 contributes to monocyte “spreading”; (3) both CCR1 and CCR5 support transendothelial chemotaxis towards CCL5. These observations suggest that the two receptors have specialized roles in monocyte recruitment. Table 3 contains a summary of chemokine molecules involved in monocyte recruitment.

Table 3. Chemokines receptors involved in monocyte trafficking.

Chemokine receptor Ligand
CCR1 (CD191) CCL3 (MIP-1a), CCL5 (RANTES), MCP2 (CCL8)
CCR2 (CD192) CCL2 (MCP1), CCL7 (MCP3), CCL12 (MCP5)
CX3CR1 CX3CR1
CCR5 CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES)
CCR6 CCL20 (MIP-3a)
CCR7 CCL19 (MIP-3b)
CCR8 CCL1
CXCR2 CXCL1 (GROa), CXCL2 (GROb), CXCL3 (GROg), MIF
CXC; cysteine-any amino acid-cysteine, CXCR; CXC chemokine receptor, CCL; cysteine-cysteine chemokine ligand, CCR; CC chemokine receptor, MCP; monocyte chemotactic protein MIP; macrophage inflammatory protein; RANTES; regulated on activation, normal T cell expressed and secreted, MIF; macrophage migration inhibitory factor


Adhesion molecules and monocyte trafficking

Monocyte recruitment follows the general paradigm of leukocyte adhesion and trafficking, which involves rolling, adhesion, and transmigration. The migration of leukocytes, including monocytes, depends on integrins and other adhesion molecules. LY6CHi monocytes in mice express L-selectin, P-selectin glycoprotein ligand 1 (PSGL1), lymphocyte function-associated antigen 1 (LFA1, also known as αLβ2 integrin), macrophage receptor 1 (MAC1, also known as integrin αMβ2), platelet endothelial cell adhesion molecule (PECAM1), and very late antigen 4 (VLA4, also known as integrin α4β1), which all contribute to leukocyte adhesion and migration [13].

Patrolling by LY6CLow monocytes along endothelial cells in resting dermal blood vessels is mediated by the integrin LFA1. In contrast, the early recruitment of LY6CHi monocytes is not affected by LFA1 deficiency. These results suggest that the adhesion mechanisms used by distinct monocyte subsets may differ depending on the type of tissue and the inflammatory state [13]. Table 4 contains a summary of adhesion molecules involved in monocyte recruitment.

Table 4. Adhesion molecules involved in monocyte trafficking.

Adhesion molecule Ligand
L-selectin Glycoproteins, CD34, GLYCAM1 and MADCAM1
PSGL P-selectin and E-selectin
LFA1 ICAM1
MAC1 ICAM1
VLA4 VCAM1
PECAM1 Endothelial PECAM1
DNAM-1 (CD226) CD155
Abbreviations: DNAM-1, DNAX accessory molecule-1; GLYCAM1, glycosylation-dependent cell adhesion molecule 1; ICAM1, intercellular adhesion molecule 1; LFA1, lymphocyte function-associated antigen 1; MAC1, macrophage receptor 1; MADCAM1, mucosal addressin cell adhesion molecule 1; PECAM1, platelet endothelial cell adhesion molecule; PSGL1, P-selectin glycoprotein ligand 1; VCAM1, vascular cell adhesion molecule 1; VLA4, very late antigen 4.


Tools to study monocytes

Isolation

Primary human monocytes can be isolated from peripheral blood mononuclear cells (PBMCs). PBMCs can be isolated from human blood or peripheral leukopacs by density gradient centrifugation. Monocytes can be isolated from PBMCs using Invitrogen Dynabeads Untouched Human Monocytes Kit, which isolates pure and viable monocytes from PBMC by negative isolation. The kit depletes T cells, B cells, NK cells, dendritic cells, granulocytes, and erythrocytes, while the negatively isolated human monocytes are left in the sample. If cell sorting instrumentation is available, then CD14, CD64, and CD192 can be used as specific markers for human monocytes.

Culture

Primary human monocytes can be cultured in vitro; however, care must be taken because within 5 days in the presence of serum or M-CSF, cultured monocytes will start to differentiate into macrophages. Monocytes grow as a mixture of adherent and nonadherent cells, with the proportion of adherent cells dependent on the culture medium and activation stimuli (if added). If autologous serum is used, RPMI 1640 culture medium is recommend such as Gibco Advanced RPMI 1640 Medium supplemented with 5% FBS.
 

Cytokine and chemokine profiling

IL-34 and CSF-1 promote the differentiation and survival of monocytes under homeostatic conditions while they are regulated by GM-CSF during inflammation. Monocytes respond to chemokine (CCL2, CCL3, CCL5) gradients that cause them to migrate from peripheral blood cells into tissues where they differentiate into macrophages, myeloid dendritic cells, or osteoclasts depending on the stimulus. Monocytes are a source of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-8 which are essential for the innate response to pathogens. Multiplexed immunoassays provide an effective way to measure monocyte associated cytokine production in plasma, serum, or cell cultures. Invitrogen multiplex assays provide an easy to use online selection tool to custom configure multiplex panels in addition to pre-configured panels.

Table 5: Key cytokines involved in monocyte differentiation, recruitment, and secretion.

  Differentiation Recruitment Secreted
Cytokines, chemokines, growth factors CSF-1, IL-34, GM-CSF CCL2, CCL3, CCL5 IL‐1α, IL-1β, IL-6, IL-8, TNFα

Multiplex immunoassays

Species Description Analytes Catalog number
Human Immune Monitoring 65-Plex Human Panel G-CSF (CSF-3), GM-CSF, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-16, IL-17A (CTLA-8), IL-18, IL-20, IL-21, IL-22, IL-23, IL-27, IL-31, LIF, M-CSF, MIF, TNF alpha, TNF beta, TSLP, BLC (CXCL13), ENA-78 (CXCL5), Eotaxin (CCL11), Eotaxin-2 (CCL24), Eotaxin-3 (CCL26), Fractalkine (CX3CL1), Gro-alpha (CXCL1), IP-10 (CXCL10), I-TAC (CXCL11), MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MDC (CCL22), MIG (CXCL9), MIP-1 alpha (CCL3), MIP-1 beta (CCL4), IP-3 alpha (CCL20), SDF-1 alpha (CXCL12), FGF-2, HGF, MMP-1, NGF beta, SCF, VEGF-A, APRIL, BAFF, CD30, CD40L (CD154), IL-2R (CD25), TNF-RII, TRAIL (CD253), TWEAK EPX650-10065-901
Mouse Immune Monitoring 48-Plex Mouse ProcartaPlex Panel BAFF, G-CSF (CSF-3), GM-CSF, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-15/IL-15R, IL-17A (CTLA-8), IL-18, IL-19, IL-22, IL-23, IL-25 (IL-17E), IL-27, IL-28, IL-31, IL-33, LIF, M-CSF, RANKL, TNF alpha, ENA-78 (CXCL5), Eotaxin (CCL11), GRO alpha (CXCL1), IP-10 (CXCL10), MCP-1 (CCL2), MCP-3 (CCL7), MIP-1 alpha (CCL3), MIP-1 beta (CCL4), MIP-2, RANTES (CCL5), Betacellulin (BTC), Leptin, VEGF-A, IL-2R, IL-7R alpha, IL-33R (ST2) EPX480-20834-901

 

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