Monocyte Markers

Zandile Nare, PhD | 2^nd April 2025

Monocytes are myeloid cells that differentiate into macrophages and dendritic cells to orchestrate both innate and adaptive immune responses. Monocytes fulfil numerous functions in the immune system, including phagocytosing pathogens and infected cells, presenting antigens to cells of the adaptive immune system, and repairing damaged tissues by clearing away debris and promoting growth.

Monocyte cell surface markers, such as CD14, CD16, HLA-DR and CX3CR1, enable the characterization of different monocyte subsets and how monocyte populations change in in different disease states or in response to different stimuli (e.g., under compound treatment) using techniques such as IHC, ICC/IF and flow cytometry. Monocytes are involved in numerous pathological conditions including atherosclerosis, neurodegenerative diseases, and infections. Monitoring numbers of these cells is therefore important for determining short term responses to infection as well as long term, chronic inflammation.

See Monocyte Marker Antibodies

Monocyte Function
Monocyte Origins
Key Monocyte Surface Markers
CD14
CD16
CCR2

CX3CR1
HLA-DR
Mouse Monocyte Markers
Other Monocyte Markers
Monocyte Marker Antibodies
References

Monocyte Function

Leukocytes, also known as white blood cells, are a diverse group of cells that mediate immune responses and can be characterised into different groups depending on their lineage and structure.¹ Representing 10% of white blood cells in humans, monocytes are evolutionarily conserved and arise from bone marrow-derived hematopoietic stem cells.^1-4

Monocytes are defined by their ability to induce direct innate immune responses, including phagocytosis of invading pathogens, cytokine release, and antigen presentation to T cells.^2,3 In humans, monocytes are divided into the three subpopulations (Table 1 and Figure 1) depending on the cell surface expression of two key markers: CD14 and CD16.^2,3,5

Monocyte Subset	Function	Disease Involvement
Classical (CD14⁺⁺CD16^-)	These cells are released from the bone marrow in response to inflammatory stimuli (e.g., bacterial infections) and are deployed to the site of infection via chemokine gradients (e.g. CCR2).⁶ Once at the site of infection, classical monocytes phagocytose pathogens and present antigens to T cells, contributing to both innate and adaptive immune responses.⁵	Involved in diseases like atherosclerosis, where they differentiate into macrophages that accumulate lipids and contribute to plaque formation.⁵ Associated with rapid CCR2-mediated recruitment to infection sites leading to excessive TNF-α/IL-1β release which worsens organ damage in sepsis.⁷
Intermediate (CD14⁺⁺CD16⁺)	Characterised by high major histocompatibility complex (MHC) class II molecule expression, making this monocyte subset efficient antigen presenting cells (APCs).⁸	Linked to atherosclerosis progression and subsequent increased risk of cardiovascular events.⁹ Act as reservoirs for viral persistence in HIV progression, with elevated CD16+ subsets being associated with chronic immune activation.^10,11
Non-classical (CD14⁺CD16⁺⁺)	Involved in surveying the vascular endothelium and expressing high levels of CX3CR1.¹² While they are less pro-inflammatory than classical monocytes, this subset can secrete cytokines (e.g., TNFa and IL-8), to contribute to chronic inflammation.^5,12	Associated with chronic inflammation in conditions such as chronic kidney disease and neurodegenerative diseases like Alzheimer’s disease.⁵ The senescent phenotype of this subset of cells can contribute to a chronic low-grade state of inflammation known as “inflamm-aging”, which is linked to ageing and age-related disease.¹²

Table 1: Monocyte subsets and their role in health and disease

Classical monocytes are primarily implicated in acute inflammatory responses, while intermediate monocytes act as APCs and play a role in chronic inflammation. On the other hand, non-classical monocytes play a role in vascular surveillance and chronic inflammation. Each subpopulation plays a dual role in health and disease, contributing to both immune defence and pathogenesis depending on the context (e.g., bacterial or viral infection, tissue injury, chronic inflammation, cancer, and autoimmunity).^5,9 Understanding the function of these monocyte subsets is vital for improved disease knowledge and for developing efficacious therapeutic interventions.^2,9

Monocyte Origins

The bone marrow acts as the primary reservoir from which monocytes are released into the circulatory system. From there, monocytes and other leucocytic cells can travel to sites of tissue damage and infection. Under homeostatic conditions, circulating monocytes can enter and remain in the spleen, which acts as a second reservoir for these cells.^4,13 Importantly, leukocytes, including monocytes, can also exist in a compartment known as the marginal pool in close proximity to the endothelial lining of blood vessels in areas of reduced blood flow.^2,14

Adhesion molecules and chemokine receptors are vital for regulating monocyte margination in tissues like the lungs.⁴ Monocytes in this pool loosely adhere to the endothelium,^2,15 from which they can be rapidly deployed to sites of injury or inflammation, and/or in response to stressors such as exercise.^2,15,16 This rapid mobilisation allows for monocytes to extravasate into tissues where they differentiate into macrophages to perform they immune functions.^2,4

Key Monocyte Surface Markers

Following their discovery, monocytes were initially characterised by their function and morphology (e.g., classical subsets are the largest, while intermediate and non-classical monocytes display a polarised and spherical morphology, respectively). However, disease processes can alter these features and affect accurate identification of monocyte populations.2 As a result, significant efforts have been made to define strict criteria for monocyte identification using cell surface molecules expressed on these cells. As such, with the application of monoclonal antibodies, flow cytometry, and transcriptomics, it is now possible to clearly define, isolate and quantify monocyte subsets based on differential expression of the key surface markers discussed below.

CD14

CD14, a glycoprotein expressed on the surface of all monocyte subsets and macrophages, plays a key role in recognising microbial pathogens, particularly lipopolysaccharide (LPS), and ringing the proverbial alarm to stimulate innate immune responses. CD14 also acts as a co-receptor with Toll-like receptor 4 (TLR4) to regulate LPS detection and stimulate cytokine production (e.g., TNFa, IL-6, and IL-8).¹⁷

Anti-CD14 antibodies can be reliably used to identify monocytes, but use with other markers (e.g., CD16) can provide more detailed insights regarding specific monocyte subsets. In addition, the role of CD14 in coordinating immune responses make it an important marker to study infection biology and immunotherapy.^6,8

Schematic of an artery, showing endothelium as the layer closest to the lumen

Figure 1: Flow cytometry dot plot showing three widely accepted human monocyte subsets in peripheral blood mononuclear cells (PBMCs) taken from healthy adults based on CD14 and CD16 expression. Rectangular borders define the classical-, intermediate-, and non-classical monocytes.

Edited and reproduced under Creative Commons 4.0 CC-BY from Cormican, S. & Griffin, M. D. Human Monocyte Subset Distinctions and Function: Insights From Gene Expression Analysis. Front. Immunol. 11, 1070 (2020).³

CD16

Acting as a receptor for the Fc portion of IgG antibodies, CD16 (FcγRIII) is expressed on classical and intermediate monocytes. In addition, CD16 is essential for promoting antibody-dependent cellular cytotoxicity (ADCC), allowing CD16-expressing monocytes to kill antibody-coated target cells.¹⁸ CD16 can be used together with CD14 in flow cytometry experiments to distinguish between classical (CD14⁺⁺CD16^-), intermediate (CD14⁺⁺CD16⁺), and non-classical (CD14⁺CD16⁺⁺ monocyte subpopulations (Figure 1).

Figure 2: Flow cytometry of human peripheral blood cells stained with PerCP-Cyanine 5.5-conjugated Anti-CD14 Antibody [MEM-15] (A121927).

Figure 3: Flow cytometry of human peripheral blood cells stained with Anti-CD16 Antibody [LNK16] (A85767).

CCR2

C-C motif chemokine receptor type 2 (CCR2, also known as CD192) is a chemokine receptor expressed on classical monocytes, facilitating their recruitment to sites of inflammation by binding to CCL2 (also known as MCP-1).³ CCR2 is crucial for monocyte migration and differentiation into macrophages in tissues. CCR2 antibodies are useful for identify classical monocytes, especially when combined in a panel with CD14 and CD16 antibodies.

CX3CR1

C-X3-C motif chemokine receptor 1 (CX3CR1), a chemokine receptor expressed on non-classical monocytes, is involved in their adhesion to endothelial cells and migration into tissues. This receptor plays a role in the surveillance of the vascular wall and in the development of atherosclerosis. CX3CR1 antibodies can be used to identify non-classical monocytes, particularly when combined with CD14 and CD16.

HLA-DR

The MHC class II molecule, human leukocyte antigen - DR isotype (HLA-DR), is expressed on monocytes, macrophages, and dendritic cells. It plays a key role in antigen presentation to T cells, facilitating immune responses. HLA-DR is often used as a marker for antigen-presenting cells and can be useful in studying immune activation and tolerance.⁵

Figure 4: IHC of human spleen stained with Anti-HLA-DR Antibody [ARC0518] (A8814).

Figure 5: Flow cytometry of human peripheral blood cells stained with Anti-HLA-DR Antibody [MEM-12] (FITC) (A85809).

Mouse Monocyte Markers

Mouse monocyte markers also enable precise identification of subset heterogeneity (i.e., classical (Ly6C^hiCD43^lo), intermediate (Ly6C^midCD43^mid), and non-classical (Ly6C^lowCD43^hi)) using flow cytometry. Key markers include Ly6C (homologous to human CD14/CD16) for subset discrimination, CCR2 (classical monocyte recruitment), and CX3CR1 (non-classical vascular surveillance).^1,4 Additional markers like CD115 (CSF1R) and CD11b (Integrin αM) aid in functional studies of bone marrow egress and tissue migration.^1,5 Antibodies against F4/80 (EMR1) and CD64 (FcγRI) further distinguish monocytes from macrophages.^1,4 These tools, combined with transgenic models, facilitate research into inflammation, infection, and cancer, bridging murine and human monocyte biology.

Other Monocyte Markers

Beyond the key markers, monocytes express additional surface receptors, adhesion molecules, and regulatory proteins that define their functional diversity. Surface receptors such as CD64 (FcγRI) and TLR4 can be used to assess pathogen-sensing capacity by flow cytometry, while adhesion/migration markers like CD11b and CD62L can be quantified using immunofluorescence to study endothelial interactions.^1,5 Cytokine receptors like CCR5 and IL-10R can be profiled via intracellular staining or RNA-seq to map inflammatory responses. Activation markers (CD86, CD69) and proliferation markers (Ki-67) are often analysed using flow cytometry or western blotting in studies of immune activation.¹² Moreover, PU.1 and IRF8 are transcriptional markers that can be used to dissect differentiation pathways.¹ These markers are critical for understanding monocyte roles in diseases with techniques tailored to experimental aims, from single-cell RNA sequencing for subset heterogeneity to functional assays like phagocytosis.