An overview of microglia subtypes and markers

Microglia are the immune effector cells of the central nervous system (CNS) and exist in three distinct forms that serve very different functional roles in the central nervous system.

Amoeboid microglia

Amoeboid microglia are associated with the developing CNS. In rats it has been shown that amoeboid microglia appear late in gestation and disappear soon after birth (Ling et al., 1980; Dalmau et al., 1997).

These cells exhibit a round cell body, possess pseudopodia and thin filopedia-like processes and contain numerous lysosomes; all traits indicative of a motile phagocytic phenotype.

During the post-natal period amoeboid microglia are believed to play a role in tissue histogenesis through the removal of inappropriate and superfluous axons (Innocenti et al., 1983; Marin-Teva et al., 2004) and through the promotion of axonal migration and growth (Polazzi & Contestabile 2002).

Ultimately, amoeboid microglia grow long crenulated processes and transform into ramified microglia found in the adult CNS (Ling 1979; Kaur & Ling 1991).

Ramified microglia

Ramified microglia are present in abundance in the brain parenchyma and constitute approximately 10–20% of the total population of glial cells in the adult (Vaughan & Peters 1974; Banati 2003).

These small round cells comprise numerous branching processes and possess little cytoplasm. In the adult brain, the resident population of ramified microglia is maintained through local cell division and through the recruitment of circulating peripheral blood monocytes (Lawson et al., 1992).

Classically, ramified microglia were considered to be inactive under physiological conditions, however, it is now known that microglia exhibit pinocytotic activity and localized motility (Booth & Thomas 1991; Thomas 1992; Fetler & Amiforena 2005).

In this respect, it has been suggested that ramified microglia contribute to metabolite removal and to the clearance of toxic factors released from injured neurons (Fetler & Amigorena 2005).

A study has even demonstrated that microglia have the propensity to transform into neurons, astrocytes or oligodendrocytes (Yokoyama et al., 2004). Hence, ramified microglia may represent a unique population of multi-potent stem cells in the adult CNS, which strongly implicates microglia in CNS repair.

Learn more about the functions of glia in the CNS.

Reactive microglia

In response to injury or pathogen invasion, quiescent ramified microglia proliferate and transform into active 'brain macrophages' otherwise known as reactive microglia (Kreutzberg 1996; Stence et al., 2001).

Microglial proliferation can be studied using antibodies raised against nuclear antigens such as Ki67 (Schluter et al., 1993; Morgan et al., 2004) or proliferating cell nuclear antigen (PCNA) (Kato et al., 2003) or by bromodeoxyuridine (BrdU) staining (Morgan et al., 2004).

Reactive microglia are rod-like, devoid of branching processes and contain numerous lysosomes and phagosomes. The reactive cell form represents a population of macrophages, which are associated with brain injury and neuroinflammation.

Following a damaging event, reactive microglia accumulate at the site of injury (Giordana et al., 1994; Dihne et al., 2001; Eugenin et al., 2001) where they play a neuroprotective role phagocytosing damaged cells and debris.

In acute lesions the peak of microglial activation occurs 2-3 days post insult, but if the pathological stimulus persists microglial activation continues (Banati 2003).

Reactive microglia expresses MHC class II antigens and other surface molecules necessary for antigen presentation including CD40, B7 and ICAM-1 (Streit et al., 1989; Benveniste et al., 2001).

Consequently, microglia are considered to be the most potent antigen presenting cells in the CNS. Like macrophages, reactive microglia secrete a number of inflammatory mediators, which serve to orchestrate the cerebral immune response.

Factors secreted include superoxide (Colton & Gilbert 1987; Si et al., 1997; Spranger et al., 1998), nitric oxide (Kingham et al., 1999), prostanoids (Minghetti & Levi 1995; Pyo et al., 1999; Pinteaux et al., 2002), glutamate (Piani & Fontana 1994; Kingham et al., 1999), quinolinic acid (Espey et al., 1997; Guillemin et al., 2003), cathepsins (Kingham & Pocock 2001), matrix metalloproteinases (Jourquin et al., 2003), interleukins (Kim et al., 2005), monocyte chemotactic protein-1 (Kim et al., 2005), tumor necrosis factor (Combs et al., 2001; Taylor et al., 2005), interferon-gamma (Suzuki et al., 2005), tissue plasminogen activator (Flavin et al., 2000) and soluble FAS ligand (Ciesielski-Treska et al., 2001; Taylor et al., 2005).

A number of neurological disorders including Alzheimer's disease (McGeer & McGeer 1995, 1996; Barger & Harmon 1997), multiple sclerosis (Diemel et al., 1998) and delayed neuronal death occurring after ischaemia (Lees 1993; Tikka & Koistinaho 2001) are associated with chronic microglial activation.

In these instances the persistent activation of microglia accompanied by the sustained secretion of inflammatory mediators is thought to have a deleterious effect on neuronal function and survival, thereby exacerbating disease processes.

Identification of microglia using antigenic markers

A paucity of ​specific microglial-only antigens has hindered microglial identification. Markers used for the detection of microglia are also present in macrophage, since both cell types exhibit the same lineage.

Microglial identification is often accomplished using flow cytometry as it enables the differences in antigen expression levels to be reliably quantified. Ramified parenchymal microglia have been demonstrated to possess the phenotype CD11b+, CD45low (Ford et al., 1995; Becher & Antel 1996), whilst other CNS macrophages and peripheral macrophages exhibit the phenotype CD11b+, CD45high (Figures 1 and 2).


Figure 1: Expression of CD45 by microglia.

CD45 expression by microglia (mic) extracted from 5 day old rat neonates as previously described (Kingham et al., 1999; Hooper et al., 2005). Microglia were isolated and left in culture for 24 hours.

The cells were subsequently harvested, fixed, then analyzed by flow cytometry using anti-CD45 (ab8216). The labeled cells are represented by the black shaded populations, whereas the unlabeled cells are depicted by the grey line (%:% of cells in M1 or M2 region, MFI: mean fluorescence intensity).


Figure 2: Expression of CD45 by peritoneal macrophages

​Extraction and staining were performed as on Figure 1. Labeled cells are represented by the black shaded populations, whereas the unlabeled cells are depicted by the grey line.


​​​Microglia can also be detected immunologically using antibodies raised against a number of macrophage-specific antigens. The downside of these procedures however, is that they fail to distinguish microglia from macrophages.

The OX-42 antibody(Graeber et al., 1989; Kingham et al., 1999; illustrated in Figure 3) recognizes the CR3 complement receptor (CD11b/CD18) expressed by rat or mouse microglia. Clone F4/80 binds a 160 kDa glycoprotein on murine ramified microglia (Perry et al., 1985).

Alternatively CD68 is a lysosomal protein and can be used to stain microglia (Graeber et al., 1990; Slepko & Levi 1996) as shown in Figure 4. High levels of CD68 expression are associated with macrophages (Figure 5) and activated microglia, whilst low levels of expression are associated with quiescent ramified microglia (Graeber et al., 1990; Slepko & Levi 1996; Kingham et al., 1999).


Figure 3: Expression of CD11b by microglia

​​CD11b expression by microglia (mic) extracted from 5 day old rat neonates as previously described (Kingham et al., 1999; Hooper et al., 2005).

Microglia were isolated and left in culture for 24 hours. The cells were subsequently harvested, fixed, then analyzed by flow cytometry using the OX-42 antibody. Labeled cells are represented by the black shaded populations, whereas unlabeled cells are depicted by the grey line (%:% of cells in M1 or M2 region, MFI: mean fluorescence intensity).​


Figure 4: Expression of CD68 by microglia

​​​CD68 expression by microglia (mic). Cells were extracted from 5 day old rat neonates as previously described (Kingham et al., 1999; Hooper et al., 2005). 

Microglia were isolated and left in culture for 24 hours. The cells were subsequently harvested, fixed then analyzed by flow cytometry using anti-CD68 (ED-1) antibodies. Labeled cells are represented by the black shaded populations, whereas the unlabeled cells are depicted by the grey line (%: % of cells in M1 or M2 region, MFI: mean fluorescence intensity).


Figure 5: Expression of CD68 by peritoneal macrophages


Identification of microglia can also be achieved using Rio Hortega's original silver carbonate staining technique or using lectin staining (Taylor et al., 2002). Lectins are carbohydrate binding proteins that label microglia through the recognition of glycoproteins containing terminal alpha-D-galactose residues (Streit & Kreutzberg 1987).

Furthermore, microglia cells can be identified using antibodies raised against the intermediate protein vimentin (Graeber et al., 1988; Slepko & Levi 1996) or by using acetylated low-density lipoprotein (LDL) conjugated to a fluorescent tag, which enables the labeling of LDL receptors. (Giulian & Baker 1986; Paresce et al., 1997). 


Learn more about glia

The functions of glia in the CNS

Radial glia cell markers and their major functions

The role of glia in demyelinating diseases pathway


References

Written by Claudie Hooper and Jennifer Pocock.

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J.M. Pocock is at Cell Signalling Laboratory, Department of Neuroinflammation, Institute of Neurology, University College London, 1 Wakefield Street, London, WC1N 1PJ.

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