All tags Neuroscience An overview of microglia subtypes and markers

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.

  • Barger SW, Harmon AD (1997). Microglial activation by Alzheimer amyloid precursor protein and modulation by apolipoprotein E. Nature 388, 878–881.
  • Banati R (2003). Neuropathological imaging: in vivo detection of glial activation as a measure of disease and adaptive change in the brain. Brit. Med. Bul. 65, 121–131.
  • Becher B, Antel JP (1996). Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia. Glia 18, 1–10.
  • Benvenisten EN, Nguyen VT, O'Keefe GM (2001). Immunological aspects of microglia: relevance to Alzheimer’s disease. Neurochem. Internat. 39, 381–391.
  • Booth PL, Thomas WE (1991). Evidence for motility and pinocytosis in ramified microglia in tissue culture. Brain Res. 548, 163–171.
  • Ciesielski-Treska J, Ulrich G, Taupenot L, Chasserot-Golaz S, Zwiller J,  Revel MO, Aunis D, Bader MF (2001). Mechanisms underlying neuronal death induced by chromogranin activated microglia. J. Biol. Chem. 276, 13113–13120.
  • Colton CA, Gilbert DL (1987). Production of superoxide anions by a CNS macrophage, the microglia. FEBS Lett. 223, 284–288.
  • Combs CK., Karlo C, Kao S, Landreth GE (2001). Beta-amyloid stimulation of microglia and monocytes results in TNFa dependent expression of inducible nitric oxide synthase and neuronal apoptosis. J. Neurosci. 21, 1179–1188.
  • Dalmau I, Finsen B, Tonder N, Zimmer J, Gonzalez B, Castellano B (1997). Development of microglia in the prenatal rat hippocampus. J. Comp. Neurol. 377, 70–84.
  • Diemel LT, Copelman CA, Cuzner ML (1998). Macrophages in CNS remyelination: friend or foe? Neurochem. Res. 23, 341–347.
  • Dihne M, Block F, Korr H, Topper R (2001). Time course of glial proliferation and glial apoptosis following excitotoxic CNS injury. Brain Res. 902, 178–189.
  • Espey MG, Chernyshev ON, Reinhard JF Jr, Namboodiri MA, Colton CA (1997). Activated human microglia produce excitotoxin quinolinic acid. Neurorep. 8, 431–434.
  • Eugenin EA, Eckardt D, Theis M, Willecke K, Bennett MV, Saez JC (2001). Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon gamma and tumour necrosis factor alpha. Proc. Natl. Acad. Sci USA. 98, 4190–4195.
  • Fetler L, Amigorena S (2005). Brain under surveillance the microglia patrol. Science. 309, 392–393.
  • Flavin MP., Zhao G, Ho LT (2000). Microglia tissue plasminogen activator (tPA) triggers neuronal apoptosis in vitro. Glia. 29, 347–354.
  • Ford AL, Goodsall AL, Hickey WF, Sedgwick JD (1995). Normal ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. J. Immunol. 154, 4309-4321.
  • Giordana MT, Attanasio A, Cavalla P, Migheli A, Vigliani MC, Schiffer D (1994). Reactive cell proliferation  and microglia following injury to the rat brain. Neuropathol. Appl. Neurobiol. 20, 163–174.
  • Glenn JA., Booth PL, Thomas WE (1991). Pinocytic activity in ramified microglia. Neurosci. Lett. 123, 27–31.
  • Graeber MB, Streit WJ, Kreutzberg GW (1988). The microglial cytoskeleton: vimentin is localiased within activated cells in situ. J. Neurocytol. 17, 573–580.
  • Graeber MB, Banati RB, Streit WJ, Kreutzberg GW (1989). Immunophenotypic characterisation of rat brain macrophages in culture. Neurosci. Lett. 103, 241–246.
  • Graeber MB., Streit WJ, Kiefer R, Schoen SW, Kreutzberg GW (1990). New expression of myelomonocytic antigens by microglia and perivascular cells following lethal motor neurone injury. J. Neuroimmunol. 27, 121–131.
  • Guilian D, Baker TJ (1986). Characterisation of amoeboid microglia isolated from developing mammalian brain. J. Neurosci. 6, 2163–2178.
  • Guillemin GJ, Brew BJ (2004). Microglia, macrophages and pericytes: a review of function and identification. Leukoc. Biol. 75, 388–397.
  • Hooper C, Taylor DL, Pocock JM (2005). Pure albumin is a potent trigger of calcium signalling and proliferation in microglia but not macrophages or astrocytes. J. Neurochem. 92, 1363–1376.
  • Innocenti GM, Clarke S, Koppell H (1983). Transitory macrophages in the white matter of the developing visual cortex. II. Development and relations with axonal pathways. Dev. Brain Res. 11, 55–66.
  • Jourquin J, Tremblay E, Decanis N, Charton G, Hanessian S, Chollet AM, Le Diguardher T, Khrestchatisky M, Rivera S (2003). Neuronal activity-dependent increase of net matrix metalloproteinase activity is associated with MMP-9 neurotoxicity after kainate. Eur. J. Neurosci. 18, 1507–1517.
  • Kato H, Takahashi A, Itoyama Y (2003). Cell cycle protein expression in proliferating microglia and astrocytes following transient global ischemia in the rat. Brain Res. Bull. 60, 215–221.
  • Kaur C, Ling EA (1991). Study of the transformation of amoeboid microglial cells into microglia labelled with the isolectin Griffonia simplicifolia in postnatal rats. Acta. Anat (Basel). 142, 118–125.
  • Kim OS., Lee CS, Kim HY, Joe EH, Jou I (2005) Characterisation of new microglia-like cells obtained from neonatal rat brain. Biochem. Biophys. Res. Commun. 328, 281–287.
  • Kingham PJ, Pocock JM (2001). Microglial secreted cathepsin B induces neuronal apoptosis. J. Neurochem. 76, 1475–1484.
  • Kingham PJ, Cuzner ML, Pocock JM (1999). Apoptotic pathways mobilized in microglia and neurones as a consequence of chromogranin A-induced microglial activation. J. Neurochem. 73, 538–547.
  • Kreutzberg GW (1996). Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, 312–318.
  • Lawson LJ, Perry VH, Gordon S (1992). Turnover of resident microglia in the normal adult mouse brain. Neurosci. 48, 405–415.             
  • Lees GJ (1993). The possible contribution of microglia and macrophages to delayed neuronal death after ischaemia. J. Neurol. Sci. 114, 119–122.
  • Ling EA (1979) Transformation of monocytes into amoeboid microglia and into microglia in the corpus callosum of postnatal rats, as shown by labelling monocytes by carbon particles. J. Anat. 128, 847–858.
  • Ling EA, Penney D, Lebond CP (1980). Use of carbon labelling to demonstrate the role of blood monocytes as precursors of the ‘amoeboid cells’ present in the corpus collosum of postnatal rats. J. Comp. Neurol. 193, 631–657.
  • McGeer PL, McGeer EG (1995). The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res. Rev. 21, 195–218.
  • McGeer PL, McGeer EG (1996). Anti-inflammatory drugs in the fight against Alzheimer’s disease. Ann. N. Y. Acad. Sci. 777, 213–220
  • Marin-Teva JL, Dusart I, Colin C, Gervais A, van Rooijen N, Mallat M (2004). Microglia promote the death of developing purkinje cells. Neuron 41, 535–547.
  • Minghetti L, Levi G (1995). Induction of prostanoid biosynthesis by bacterial lipopolysaccharide and isoproterenol in rat microglial cultures. J. Neurochem. 65, 2690–2698.
  • Morgan SC,  Taylor DL, Pocock JM (2004). Microglia release activators of neuronal proliferation mediated by activation of mitogen-activated protein kinase, phosphatidylinositol-3-kinase/AKT and delta-Notch signalling cascades. J. Neurochem. 90, 89–101.
  • Nimmerjahn A, Kirchhoff F, Helmchen F (2005). Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308, 1314–1318.
  • Paresce DM., Chung H, Maxfield R (1997). Slow degradation of aggregates of the Alzheimer’s disease amyloid b protein by microglial cells. J. Biol. Chem. 272, 29390–29397.
  • Perry VH, Hume DA, Gordon S (1985). Immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain. Neurosci. 15, 313–326.
  • Piani D, Fontana A (1994). Involvement of the cystine transport system xc in the macrophage induced glutamate dependent cytotoxicity to neurones. J. Immunol. 152, 3578–3585.
  • Pinteaux E, Parker LC, Rothwell NJ, Luheshi GN (2002). Expression of interleukin 1 receptors and their role in interleukin 1 actions in murine microglial cells. J. Neurochem. 83, 754–763.
  • Polazzi E, Contestabile A (2004). Reciprocal interactions between microglia and neurons: from survival to neuropathology. Rev. Neurosci. 13, 221–242.
  • Pyo H, Joe E, Jung S, Lee SH, Jou I (1999). Gangliosides activate cultured rat brain microglia. J. Biol. Chem. 274, 34584–34589.
  • Schuter C, Duchrow M, Wohlenberg C, Becker MH, Key G, Flad HD, Gerdes J (1993). The cell proliferation-associated antigen of antibody ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle maintaining proteins. J. Cell Biol. 123, 513–522.
  • Si QS, Nakamura Y, Kataoka K (1997). Albumin enhances superoxide production in cultured microglia Glia. 21, 413–418.
  • Slepko N, Levi G (1996). Progressive activation of adult microglial cells in vitro. Glia 16, 241–246.
  • Spranger M, Kiprianova I, Krempien S, Schwab S (1998). Reoxygenation increases the release of reactive oxygen intermediates in murine microglia. J. Cereb. Blood Flow Met. 18, 670–674.
  • Stence N, Waite M, Dailey E (2001). Dynamics of microglial-activation: a confocal time-lapse analysis in hippocampal slices. Glia 33, 256–266.
  • Streit WJ. Kreutzberg GW (1987). Lectin binding by resting and reactive microglia. J. Neurocytol. 16, 249–260.
  • Suzuki Y, Claflin J, Wang X, Lengi A, Kikuchi T  (2005). Microglia and macrophages as innate producers of interferon-gamma in the brain following infection with Toxoplasma gondi. Int. J. Parasitol. 35, 83–90.
  • Taylor DL., Diemel LT, Cuzner ML, Pocock JM (2002). Activation of group III metabotropic glutamate receptors underlies microglial reactivity and neurotoxicity following stimulation with chromogranin A, a peptide up-regulated in Alzheimer’s disease. J. Neurochem. 82, 1179–1191.
  • Taylor DL, Jones F, Kubota ES, Pocock JM (2005). Stimulation of microglial metabotropic glutamate receptor mGlu2 triggers tumor necrosis factor alpha-induced neurotoxicity in concert with microglial-derived Fas ligand. J. Neurosci. 25, 2952–2964.
  • Thomas WE (1992). Brain macrophages: evaluation of microglia and their functions. Brain Res. Rev. 17, 61–74.
  • Tikka TM, Koistinaho JE (2001). Minocycline provides neuroprotection against N-methyl-D-aspartate neurotoxicity by inhibiting microglia. J. Immunol. 166, 7527–7533.
  • Vaughan DW, Peters A (1974). Neuroglial cells in the cerebral cortex of rats from young adult to old age: an electron microscopy study. J. Neurocytol. 3, 405–429.
  • Yokoyama A, Yang L, Itoh  S, Mori K, Tanaka J (2004). Microglia, a potent source of neurons, astrocytes and oligodendrocytes. Glia 45, 96–104.

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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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