Mini-Review: CD68 — A Macrophage and Monocyte Marker

Overview

CD68 is a heavily glycosylated type I transmembrane glycoprotein. It is considered a member of both the lysosome-associated membrane glycoprotein (LAMP) family, due to its predominant localization to the lysosomes and endosomes, and the scavenger receptor family, which functions to clear debris and promote phagocytosis (Chistiakov et al. 2017).

CD68 is extensively used as a marker of myeloid cells, particularly macrophages and monocytes, due to its high expression in these populations (Wei et al. 2023). Bio-Rad offers a wide range of CD68 antibodies in a variety of formats and sizes for various applications. For applications such as flow cytometry, CD68 antibodies are available conjugated to a range of fluorescent labels, such as the Alexa Fluor dyes, FITC, and PE.

CD68 Expression

Human CD68 is a 110 kD protein, comprised of 354 amino acids (Holness and Simmons 1993). The murine homologue of CD68, macrosialin, shares 72% identity and 80.6% similarity in the amino acid sequence to human CD68 (Chistiakov et al. 2017).

Bio-Rad’s range of CD68 antibodies includes those against human, mouse, and rat forms of the protein, allowing you to design appropriate experiments for your target species. The collection includes three clones for the detection of the human protein (Y1/82A, 514H12, and KP1), one mouse clone (FA-11), and one rat clone (ED1).

Anti-CD68 antibodies can be used to stain a variety of cell types in your experiments. Cells of the mononuclear phagocyte lineage are high expressers of the CD68 protein. This family includes monocytes, macrophages, Kupffer cells, osteoclasts, microglia, tissue histiocytes, and myeloid dendritic cells (DCs) (Chistiakov et al. 2017).

It’s important to note that, while CD68 is best known as a macrophage marker, these antibodies can also be used alongside additional markers to identify other cell types.

Gottfried et al. (2008) found that CD68 staining intensity in primary fibroblasts and endothelial cells was comparable to macrophages, depending on the clone and application utilized. Additionally, CD68 staining in lymphocytes was also observed, albeit to a much lesser extent. This suggests that the potential uses of anti-CD68 antibodies are much more diverse than previously thought.

While CD68 can be found to some degree on the cell surface, it is predominantly located in the endosomal/lysosomal compartment within the cell. You will need to keep this in mind when designing your experiments as your sample preparation protocols may need to be adjusted accordingly.

Bio-Rad provides a protocol for the direct staining of intracellular CD68 for flow cytometry using Leucoperm Accessory Reagent. Additionally, for immunohistochemical detection of CD68 on paraffin sections, heat-mediated antigen retrieval is required. We recommend using a citrate buffer, pH 6.0, for this.

CD68 in Cancer Research

Now that we have established the basics of CD68 expression, we can dive deeper into the specific research areas in which this marker is typically used.

For example, CD68 is the most commonly used marker to identify tumor-associated macrophages (TAMs) (Song et al. 2023). TAMs are a highly abundant cell type in solid tumors that can induce T-cell apoptosis and recruit Tregs to suppress antitumor activity. Additionally, they have been shown to promote tumor growth via the induction of angiogenesis (Kusmartsev and Gabrilovich 2005; Qiao et al. 2023; Wang et al. 2017).

High expression levels of CD68 in tumors is associated with larger tumor size, higher tumor grade, and poor clinical outcomes in various cancers. Zhang et al. (2022) observed a close correlation between CD68 levels and immune infiltrates in the TME, and subsequently suggested that CD68 could be a promising future target for immunotherapy options.

If you are interested in studying TAMs in your own research, Bio-Rad offers an extensive range of cancer antibodies, proteins, and reagents that can be used in combination with CD68 to explore key areas of tumor biology.

CD68 in Bone Research

As previously mentioned, CD68 is highly expressed in osteoclasts, the specialized myeloid cells in bones. Thus, CD68 is also a significant player in the field of bone research.

While the skeletal system may appear to be a static structure, it is, in reality, a dynamic organ that is continually undergoing a process of bone breakdown — a process known as bone resorption — by osteoclasts and bone formation by osteoblasts. Under normal physiological conditions, these processes are balanced to avoid excessive bone loss or abnormal bone growth (Hadjidakis and Androulakis 2006).

To analyze the significance of CD68 in osteoclasts, Ashley et al. (2011) generated mice lacking CD68 expression and assessed the effect on their bones. They found that the deletion of CD68 led to significantly increased trabecular bone, indicating a defect in bone resorption, which was reinforced by in vitro data showing that osteoclasts from these mice were able to resorb a significantly smaller area of bone slices than their wildtype counterparts.

In this study, anti-CD68 antibodies were used in western blotting, flow cytometry, and immunofluorescent microscopy, demonstrating the breadth of value provided by this marker.

CD68 in Inflammation

One key application in which CD68 staining is often utilized is in the detection of inflammation due to its role as a macrophage marker, and the infiltration and accumulation of macrophages during inflammation (Pahwa et al. 2023; Bruun et al. 2006).

While CD68 is used as a marker of inflammation, its specific role in the immune response remains uncertain. Classically, members of the scavenger family were identified to play a key role in the binding and degradation of oxidized low-density lipoproteins (oxLDL), a marker of oxidative stress (Zani et al. 2015).

It is now known that scavenger receptors can play a much more diverse role in immunity. For example, they can recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) to trigger the production of anti-inflammatory cytokines and enable the phagocytosis of pathogens (Taban et al. 2022).

However, Song et al. (2011) used a mouse in which the CD68 gene was genetically deleted and found that the ability of macrophages to take up oxLDL, respond to PAMPs and DAMPS, and phagocytose pathogens was not significantly different to that of wildtype macrophages. Therefore, the exact role of CD68 in innate immunity remains unknown.

Interestingly, in the same study, a trend was observed in which mononuclear phagocytes lacking CD68 displayed enhanced antigen presentation abilities to CD4+ T cells. This may indicate that CD68 negatively regulates antigen presentation and, ultimately, T cell activation.

To enable the further study of CD68’s impact on the adaptive immune response, Bio-Rad offers an extensive range of T cell-related antibodies, including those targeting the T cell receptor, co-receptors, and cell subtype-specific markers.

Overall, previous studies present an interesting role for CD68 in various scientific areas. However, there are still many questions left unanswered about the function of the protein.

If you feel inspired to utilize this valuable marker in your research, discover Bio-Rad’s range of CD68 antibodies.

References

  • Ashley JW et al. (2011). Genetic ablation of CD68 results in mice with increased bone and dysfunctional osteoclasts. PLoS One 6, e25838. 
  • Bruun JM et al. (2006). Diet and exercise reduce low-grade inflammation and macrophage infiltration in adipose tissue but not in skeletal muscle in severely obese subjects. Am J Physiol Endocrinol Metab 290, E961–967.
  • Chistiakov DA et al. (2017). CD68/macrosialin: not just a histochemical marker. Lab Invest 97, 4–13.  
  • Gottfried E et al. (2008). Expression of CD68 in non-myeloid cell types. Scand J Immunol 67, 453–463.
  • Hadjikdakis DJ and Androulakis II (2006). Bone remodeling. Ann N Y Acad Sci 1092, 385–396.
  • Holness CL and Simmons DL (1993). Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. Blood 81, 1607–1613.
  • Kusmartsev S and Gabrilovich DI (2005). STAT1 signaling regulates tumor-associated macrophage-mediated T cell deletion. J Immunol 174, 4880–4891.
  • Pahwa R et al. (2023). Chronic inflammation. Available from: StatPearls [Internet].
  • Qiao T et al. (2023). Dynamic differentiation of F4/80+ tumor-associated macrophage and its role in tumor vascularization in a syngeneic mouse model of colorectal liver metastasis. Cell Death Dis 14, 117.   
  • Song J et al. (2023). Tumor-associated macrophages: Potential therapeutic targets and diagnostic markers in cancer. Pathol Res Pract 249, 154739.
  • Song L et al. (2011). Deletion of the murine scavenger receptor CD68. J Lipid Res 52, 1542–1550.
  • Taban Q et al. (2022). Scavenger receptors in host defense: from functional aspects to mode of action. Cell Commun Signal 20, 2. 
  • Wang J et al. (2017). Tumor cells induced-M2 macrophage favors accumulation of Treg in nasopharyngeal carcinoma. Int J Clin Exp Pathol 10, 8389–8401.  
  • Wei Q et al. (2023). The markers to delineate different phenotypes of macrophages related to metabolic disorders. Front Immunol 14, 1084636
  • Zani IA et al. (2015). Scavenger receptor structure and function in health and disease. Cells 4, 178–201.
  • Zhang J et al. (2022). Role of CD68 in tumor immunity and prognosis prediction in pan-cancer. Sci Rep 12, 7844.