Microglia: gatekeepers of central nervous system immunology

Bart R. Tambuyzer,*,,1 Peter Ponsaerts, and Etienne J. Nouwen*

*Laboratory of Neurobiology and Neuropharmacology, Department of Biomedical Sciences, and Laboratory of
Experimental Haematology, Vaccine and Infectious Disease Institute (Vaxinfectio), Faculty of Medicine, University of
Antwerp, Belgium

Abstract: Microglia are perhaps the most underestimated cell type of our immune system. Not only
were immunologists unaware of their capabilities until recently, but also, some neuroscientists denied
their actual existence until the late 20th century.
Nowadays, their presence is confirmed extensively,
as demonstrated by numerous reports describing
their involvement in virtually all neuropathologies.
However, despite distinct approaches, their origin
remains a point of controversy. Although many agree
about their myeloid-monocytic ancestry, the precise
progenitor cells and the differentiation mechanisms,
which give rise to microglia in the different developmental stages of the CNS, are not unraveled yet.
Mostly, this can be attributed to their versatile phenotype. Indeed, microglia show a high morphological
plasticity, which is related to their functional state.
This review about microglia aims to introduce the
reader extensively into their ontogeny, cell biology,
and involvement in different neuropathologies. J.
Leukoc. Biol. 85: 352370; 2009.
Key Words: review immune function activation neuropathology

ORIGIN OF MICROGLIA
The developmental origin of microglia has been a subject of
debate for many years since del Rio-Hortega [3] first described
this cell type in 1932. Nowadays, most researchers support the
hypothesis that microglia are derived from myeloid-monocytic
cells and/or their hematopoietic precursors (for reviews, see
refs. [4, 5]). Following this hypothesis, microglia or their precursors should enter the CNS at a certain point and migrate and
differentiate to ultimately colonize the whole CNS parenchyma.
In contrast, others believe that microglia are derived from
neuroectodermal matrix cells [6], which are able to differentiate into microglia locally. The latter implies a common ancestry for microglia and macroglia, such as astrocytes and oligodendrocytes. In addition, some other schools support the hypothesis that microglia originate from pericytes [7] or from the
subependyma adjacent to the lateral ventricles [8].
Here, we will review in depth the two most important hypotheses for microglial origin: neuroectodermal or myeloidmonocytic. For the latter, we will also discuss migration and
differentiation of microglia (or their precursors) in the developing CNS. Although this myeloid-monocytic origin for microglia has been widely accepted now, the neuroectodermal hypothesis remains interesting from a historical perspective.

Microglial precursors
MICROGLIA: AN HISTORICAL PERSPECTIVE
The earliest report of microglia can be traced back to the late
19th century. The psychiatrist Nissl [1] encountered rod cells
in the CNS and described them as reactive glial elements with
the ability to migrate, proliferate, and perform phagocytosis.
Next, Santiago Ramon y Cajal in 1913 [2] was the first to
determine a third element in the CNS next toin that time
commonly known neurons and astrocytes, a distinction he
made based on morphological differences. However, Pio del
Rio-Hortega [3], a Spanish neuroanatomist, was the first to
divide this third element into oligodendroglia and microglia.
Using silver-staining methods and light microscopy, he described several fundamental characteristics of microglia that
are still considered to be of relevance for current research
about microglia. In his work, he concluded that microglia
originated from the invasion of mesodermal pial elements into
the nervous system, but he also speculated about blood mononuclear cells as a potential source. His novel insights, together
with the limitations of the silver-staining methods as the only
identification procedure for many decades, gave rise to a longlasting controversy about microglial ontogeny.
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Neuroectodermal precursors

The view that microglia are derived from neuroectodermal

progenitor cells has been proposed by several independent
researchers using different techniques. Skoff [9], who used a
rat model of optic nerve degeneration and optic nerve development in combination with 3H-autoradiography and electron
microscopy, was the first to detect a so called multipotential
glia cell. According to the data, these cells originated from
neuroectodermal matrix cells and not from invading mesenchymal cells. This was confirmed using the same techniques in
newborn mice, where a continuous morphological transition
was observed between glioblasts and resting (ramified)
microglia in the developing gray matter of the hippocampus
[10].

1
Correspondence: Laboratory of Experimental Haematology, Vaccine and Infectious Disease Institute (VIDI), University of Antwerp, Universiteitsplein 1,
B-2610 Wilrijk, Belgium. E-mail: bart.tambuyzer@ua.ac.be
Received June 26, 2008; revised October 23, 2008; accepted October 27,
2008.
doi: 10.1189/jlb.0608385

0741-5400/09/0085-352 Society for Leukocyte Biology

Second, others were able to identify microglia precursors in

the germinal matrix of the CNS based on lectin [Ricinus
communis agglutinin 1 (RCA-I)] or immunohistochemical
stainings [11, 12]. The glial raphe of the floor plate of the
hindbrain and spinal cord were pinpointed as the site of
microglia origin through immunohistochemical staining for lipocortin-1 (annexin-I). Disialoganglioside GD3 is another molecule described to be specific for microglia and is also expressed on neuroectoderm, oligodendrocytes, and specific neuron populations [13]. However, sharing markers does not
always imply a common ancestry.
A third series of experiments supporting the neuroectodermal origin hypothesis involved cell culture experiments using
brain tissue from different developmental stages, appropriately
discarded of blood (vessels) and meninges, from which microglia could be recovered. In this experimental set-up, microglia
were derived from their progenitors in neopallial cultures of
embryonic and newborn mice after nutritional deprivation [14].
In addition, this study was expanded by culturing the neopallial cells in Grenier hybridoma tissue-culture dishes. This led
to heterogeneous clones of astroglia and microglia, supporting
the common progenitor hypothesis [15]. The fact that microglia
and astroglia could have a common origin was also corroborated by a report about hematopoietic stem cells as a source for
both cell types in adult mice brain [16]. However, in this study,
hematopoietic stem cells were derived from bone marrow and
could have been contaminated with other progenitor cells,
which might explain their divergent differentiation potential.
Hematopoietic stem cells

The appearance of microglia in the developing brain before

onset of vascularization [17] or before the appearance of monocytes in hematopoietic tissues [18] favors hematopoietic stem
or progenitor cells to be the source of microglia in situ. Initial
attempts to demonstrate the presence of hematopoietic stem
cells in the mammalian brain were undertaken by injecting
cells of dissociated brain tissue into irradiated mice [19]. A
significant number of CFUs were found in the spleen, leading
to the conclusion that hematopoietic stem cells might colonize
the CNS during embryogenesis. However, others used the same
experimental model but washed the brains extensively prior to
cell dissociation [20]. In addition, methylcellulose cultures
were performed to detect GM-CFUs [21]. Only few colonies
were observed, leading to the conclusion that the hematopoietic stem cells recovered by Bartlett [19] were of contaminating
blood origin. In contrast, Alliot et al. [22] reported the existence of hematopoietic stem cells that can, next to differentiation into blood cell types, transform into microglia in the
developing and adult CNS of mice. Experimental evidence
demonstrated that these cells could be traced back to the yolk
sac and produced microglia already in early embryogenesis
[23]. To this extent, a subpopulation of bone marrow cells was
identified, sharing the same highly specific ion channel pattern
as microglia [24], suggesting a precursor role for these cells.
Another approach involved the use of chimeric animals.
Following irradiation, host animals were injected with bone
marrow cells expressing a different MHC class I haplotype.
However, only few microglia (-like) cells were identified within
the CNS even after a long recovery time [25]. The same setup,

combined with a model of experimental allergic encephalomyelitis (EAE) to up-regulate expression of MHC class I in CNS
immunocytes, gave rise to similar results [26]. To overcome
problems as a result of marker expression, bone marrow cells
from bacteriophage-infected transgenic mice were used as a
donor. Despite the presence of this stable marker, only few
donor-type microglia could be detected in host CNS [27].
Recently however, transplantation of GFP mice bone marrow
cells in GFP host mice revealed the presence of many GFP
microglia throughout developing and/or inflamed CNS [28, 29].
In conclusion, although some claim that microglia themselves
can serve as pluri/multipotential stem cells to form neurons,
astrocytes, and oligodendrocytes in vitro [30, 31], markers
distinguishing microglia univocally from other cell lineages in
the CNS or the peripheral system have yet to be defined.
Monocytes/macrophages as direct progenitors

According to the model of Van Furth [32], microglial origin

should be classified into the mononuclear phagocyte system.
Indeed, amoeboid and ramified microglia express the two most
important markers characteristic for this system: the FcR and
the complement receptor III (CR3) [33, 34]. In addition, amoeboid microglia are capable of performing phagocytosis in vitro
[35] and in vivo [36]. Although these characteristics constitute
the minimal requirements to be a true mononuclear phagocyte, it does not exclude a nonmonocytic origin for microglia.
Several other markers shared by microglia with monocytes
and/or macrophages have been used to sustain the monocyte
origin hypothesis: e.g., enzyme cytochemical stainings for nucleoside diphosphatase, acid phosphatase, nonspecific esterase
(NSE) [37, 38] vaults (large-size ribonuclear protein particles)
[39], and in particular, lectin staining (e.g., Griffonia simplicifolia isolectin-b4, RCA, wheat germ agglutinin, and Con A)
[40]. However, these observations do not permit us to outline a
straightforward staining for all microglia to confirm their monocytic origin. Frequently, amoeboid microglia stain intensively,
but their ramified counterparts do not express the same markers [40, 41]. Also, as described above, microglia share some
markers with astroglia and oligodendrocytes [13, 42]. In addition, microglia display specific characteristics such as proliferation in vitro [35] and in vivo (see Proliferation) and a
specific ion channel pattern [43], which are different as compared with monocytes/macrophages.
Several interesting studies performed by Ling and colleagues [44], in which colloidal carbon particles were injected
to follow blood monocytes into the developing rat CNS, demonstrated that monocytes that had ingested the carbon particles
peripherically appeared as amoeboid microglia in the developing CNS and later on as ramified cells. The same investigators repeated these experiments with rhodamine B isothiocyanate [45]. Although most of the microglia initially appear
during perinatal CNS development through recruitment of their
progenitor cells, their population is sedentary in the normal
adult CNS and is replenished only partially by monocytic/
myeloid precursors under pathological circumstances [26].
However, in some models, e.g., EAE [46] and ischemic retinopathy [29], an accelerated turnover of microglia by bone
marrow-derived cells circulating in the blood was observed. In
addition, indirect support for a monocytic origin was supplied
Tambuyzer et al. An introduction to microglia

353

by a study showing depletion of amoeboid microglia in postnatal brain after administration of glucocorticoids, which are
known to be strong immunosuppressors [47].
New concepts

An interesting recent study performing chimeric bone marrow

transplantations points out that microglia as a population balance between mitosis and apoptosis during inflammation to
control their numbers [48]. Moreover, following irradiation of
the host, they demonstrate that resident microglia are complemented with a small population of migrating microglia originating from bone marrow transplants. In addition, they demonstrated that the number of resident microglia in their chimera modelmicroglia expected not to be killed by
irradiationwas reduced up to 30% in lesion-induced inflammation and that they were severely affected in their mitotic
ability. To further demonstrate the bone marrow-derived origin
of microglia, experiments were performed in which two circulating systems of different mice were interconnected (i.e., parabiosis). In this set-up, supply of labeled cells to the recipient
mouse did not result in a contribution to the microglial population in these hosts, even after inflammation was evoked [49].
However, only when irradiation was combined with bone marrow injection, donor-derived microglia were observed in brain
tissue. The latter suggests for an experimentally induced migration of bone marrow-derived precursors after irradiation and
injection in the bloodstream as used in many other (older)
studies (see Hematopoietic stem cells and Monocytes/macrophages as direct progenitors), perhaps not truthfully mimicking
true microglial ontogeny processes. In contrast, Mildner and
colleagues [50] designate Ly-6ChiCCR2 monocytes as microglial precursors but also underscore the need for irradiation of
the brain to induce migration of microglial precursors. From
these new studies, using bone marrow/monocytes in combination with irradiation to demonstrate migration/origin of microglia, it can be concluded that caution should be taken to design
a relevant study model, as each experimental set-up currently
used might harbor several important complications as commented by Ransohoff [51].

Entry of microglial precursors in the CNS

A massive appearance of microglia is seen around birth in
different species; i.e., the so-called developmental window of
these cells [6] at a time vascularization of the CNS is already
completed. However, microglia also appear, to a lesser extent
in embryonic life [18, 23], in the avascular CNS. Moreover, the
neuroectodermal origin hypothesis would implicate their presence within the developing CNS, as suggested by McKanna
[12].
If microglia originate from a sourcein casu monocytes or
myeloid precursors outside the CNS, they would have a site
of entry at some point. The completed vascularization of the
CNS forms the perfect basis for a widespread microglial colonization. Indeed, several researchers support monocyte recruitment from the bloodstream as a basis for the expansion of
microglial cell populations [33, 39, 44]. As one can expect, the
brain endothelium plays a key role in this process by expressing adhesion molecules ICAM-1 [52] and -2 [53] as homing
receptors.
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Journal of Leukocyte Biology Volume 85, March 2009

A second possibility of microglial entrance into the CNS is

via the ventricles by passing the ventricular surface. Ventricular macrophages were also observed in the developing brain
and were associated closely with the appearance of microglia in
the corresponding nervous parenchyma [4, 54]. In addition,
one study was able to track microglial cells while passing the
ventricular surface [55].
Third, it has been suggested that microglia are derived of a
meningeal source. As del Rio-Hortega [3] described the invasion of pial elements, several others demonstrated precursors
crossing the pial surface to become microglia [56]. In addition,
a report in avian embryos suggested that microglia invade the
CNS and through proliferation, populate the entire CNS [57].

Proliferation, migration, and differentiation

Upon arrival in the CNS, microglial precursors are expected to
undergo three major processes toward becoming a fully differentiated microglial cell: proliferation from a precursor group,
migration of the expanded cells to populate different CNS
regions, and differentiation of the amoeboid microglia into their
ramified resting phenotype. Throughout these processes, microglia undergo many transformations, a sequence of events
often referred to as developmental plasticity.
Proliferation

Several molecules, among them IL-3, IL-6, GM-CSF, and

CSF-1, have been demonstrated to be potent stimuli for microglia in vitro [58 61]. For all of these cytokines, microglia
express the appropriate receptors [61]. In addition, all of the
growth factors mentioned above can be produced locally in the
CNS [62 65]. Several in vivo studies using proliferating cell
nuclear antigen [66], Ki67 [67], BrdU [68], and autoradiography [69] have confirmed the hypothesis that microglia can
proliferate in a developing brain environment.
Migration

Nowadays, it is common knowledge that microglia are capable

of migrating toward damaged neural tissue to clear the site of
injury when necessary [36, 70]. However, the mechanisms
thriving microglia to populate the CNS after arrival at entrance
gates are poorly understood. According to extensive studies
about quails by Navascues and co-workers [71], microglial
migration during development occurs in a two-step process:
tangential migration followed by a radial movement pattern.
Microglia have been shown to migrate tangentially along nerve
fibers (so-called highways) in the retina [72], through the
stratum album centrale in the optic tectum [55] and along the
developing white matter in the cerebellum [73]. At least in the
retina, this process takes place on a substratum of Muller cell
endfeet [72]. Radial migration from these highways has been
described clearly using confocal microscopy in quail retina
[74]. The latter has also been described for humans by Rezaie
and Male [66], who reported that microglial invasion advances
in radial waves along radiating cortical vessels and radial glial
fibers. They pinpointed the importance of adhesion molecules,
such as ICAM-2 and PECAM, on cerebral blood vessels and
the production of the chemokines MCP-1 and RANTES. In
addition, these migratory processes can be triggered by extenhttp://www.jleukbio.org

sive cell death necessary for CNS remodeling during development [75], although these events are not always related [76].

microglia are distributed unevenly within the mouse hippocampal regions and the cerebellum [94, 95].

Differentiation

Morphology

It is largely accepted now that microglia acquire a ramified

morphology at their final location in the CNS parenchyma [77].
This ramification process has been reported to occur simultaneously during radial migration of microglia [74]. The migratory properties of ramified microglia have also been confirmed
in vitro [78]. Observations of intermediate forms between
amoeboid and ramified microglial subpopulations further support the ramification hypothesis [33, 45, 66]. Also, during
morphological transformation, the microglial phenotype
changes drastically: CD11b, MHC I and MHC II, lectin, and
CD45 immunoreactivity decreases or disappears [17], and enzymatic activity, e.g., NSE, abolishes with the transformation of
an amoeboid microglial cell to the ramified form [41]. The
ramified resting state of microglia in the CNS is induced and
sustained via direct contact between neuronal cells and microglia involving a CD200 (neurons)CD200R (microglia) interaction. CD200, expressed constitutively by neurons and CNS
endothelium, provides an inhibitory signal for microglia [79].
In addition, several in vitro studies validated the in vivo
observations. Isolated amoeboid microglia can acquire the
resting ramified phenotype when cocultured on top of astrocytes [80] or epithelial cell layers [80, 81]. Even more, microglia can perform transmigration through a renal epithelial
monolayer in vitro, acquiring the ramified morphology under
these cells [82]. Ramification in vitro is reversible [83], mimicking the process of dedifferentiation in vivo. Soluble factors
able to induce ramification include vitamin E [84], DMSO, and
retinoic acid [35], GM-CSF and/or M-CSF/GM-CSF combinations [85 87], and conditioned media [81]. In general, it is
assumed that serum inhibits ramification [85, 88], although
some maintain the view that microglia do not ramify in absence
of serum [81]. The molecular mechanisms allowing ramification remain to be elucidated, although the formation of stableacetylated and detyrosinated microtubules seems to be necessary for the transition of amoeboid microglia into ramified cells
[89].

The term developmental plasticity describes the processes of

morphological/phenotypical transformation of amoeboid microglia into the ramified form during development (vide supra).
However, these resting ramified microglia are capable of dedifferentiating in the amoeboid form: a process often referred to
as functional plasticity [92]. Based on studies of the normal
adult brain and on pathophysiological studies, three major
differentiation states of parenchymal microglia can be distinguished [90]. Under normal circumstances, they appear as
ramified, resting microglia; upon environmental stimulation,
they become activated, nonphagocytic microglia with a
bushy appearance (for an example, see Fig. 1 in ref. [96]);
when stimuli persist, they acquire the amoeboid phenotype, a
fully functional, phagocytic microglial cell. However, the definition of ramified microglia as resting has been challenged
recently as a result of the observations that ramified microglia
can migrate in vitro on an astrocyte monolayer at 20 35 m/h
[79]; ramified microglia posses a highly dynamic plasticity with
constantly protruding and retracting branches, as demonstrated
by in vivo two-photon laser-scanning microscopy [97, 98]. In
view of the latter, microglia scan the CNS parenchyma completely every few hours [99].
Topographical differences in morphology have also been
reported for ramified microglia [33, 93]. In gray matter, their
processes usually are organized in a radiate manner. In contrast, in white matter, their cytoplasmatic extensions align
parallel or perpendicular to nerve fiber tracts. In this way, the
shape of the ramified microglia is well adapted to the architecture of the CNS region they populate. Not only does the
structure of the CNS influence the microglial morphology, but
also, the type of neuronal degeneration affects the transition of
ramified microglia (for a review, see ref. [96]). Indeed, morphological transition of ramified microglia toward a bushy
appearance is associated with anterograde axonal, and terminal
synaptic degeneration, toward a rod cell-like morphology, is
associated with dense dendritic degeneration; clusters of hyper-ramified microglia are observed around degenerating, single neurons. In this sense, the term bushy, as described above,
may cover a heterogeneous group of activated microglia morphologically designed to a specific context.

DISTRIBUTION, MORPHOLOGY, AND

MICROGLIAL MARKERS

Microglial markers
Distribution
Microglia are estimated to comprise 520% of all glial cells in
the CNS [90 92]. Assuming there are 10 times more glia than
neurons in the CNS, there are as much microglia as there are
neurons [93]. However, there seem to be regional differences in
the distribution of microglia in the CNS, as they appear more
in the gray than in the white matter [3, 33, 91]. In addition,
microglia are sparser in regions such as the hippocampus and
the dorsal thalamus in the developing CNS [33]. In the adult
mouse, the highest microglial densities are found in the hippocampal formation, the olfactory telencephalon, portions of
the basal ganglia, and the substantia nigra (SN) [90]. Moreover,

For a long time, microglial characterization was limited to

silver carbonate staining [3]. The difficulty in describing a
uniform characterization procedure for microglia lies in their
interchangeable phenotype and concomitant differences in expression profile. The following table summarizes a large number of common membrane and intracellular proteins expressed
by primary microglia in vivo and in vitro; proteins expressed
constitutively during all differentiation stages are printed in
bold, and the other proteins are associated with microglial
activation (Table 1). The functional relevance of these proteins will be discussed further in the context of microglial
activation and neuropathology.
Tambuyzer et al. An introduction to microglia

355

TABLE 1.

Microglial Markers

Enzyme markers:

References

NSE (cytoplasm)
Acid phosphatase (lysosomes)
Nucleoside diphosphatase (plasma membrane)
Thiamine pyrophosphatase (plasma membrane)
Nonenzymatic
membrane
markers:
CD4
CD11a
CD11b
CD11c
CD13
CD14
CD16
(FcRIII)
CD18
CD24
CD32
(FcRII)
CD34
CD36
CD107a
CD163
CD200R
MHC I
MHC II
lectins
F4/80
TLR19a
LN-1
LN-3
GLUT-1
GLUT-5
Nonenzymatic
intracellular
markers:
2-Microglobulin
GD3
laminin
Vimentin
Iba1
CD68

[100]
[101]
[37]
[102]

References
[100]
[103]
[104]
[105]
[106]
[104]

F4/80
CD40 (TNFR)
CD44
CD45
CD54
CD58

[122]
[123]
[123]
[124]
[125]
[126]

[34]
[107]
[108]

CD64 (FcRI)
CD80 (B7-1)
CD86 (B7-2)

[34]
[127]
[127]

[34]
[109]
[110]
[111]
[112]
[113]
[114]
[115]
[116]
[33]
[117]
[118]
[119]
[120]
[121]

CD87
CD95/CD95L
CD106
EP2 receptor
opioid receptors
acetylated LDL receptor
cytokine/chemokine receptors
cannabinoid receptors
CRC5a
CRC1q
purinogenic receptors
benzodiazepine receptors
mannose receptors
vitronectin receptor
PPAR-

[128]
[129]
[130]
[131]
[132]
[35]
[133]
[116]
[134]
[135]
[136]
[137]
[138]
[139]
[140]

References
[141]
[23]
[104]
[142]
[143]
[144]

calprotectin
substance P
vaults
fibronectin
TLR19a

[87]
[145]
[39]
[104]
[117]

TLR1, -2, -4, -5, -6, and -9 are expressed extracellularly, and TLR3, -7,
-8, and -9 were found to be restricted to the intracellular compartments. LN-1,
Laminin-1; GLUT-1, glucose transporter-1; CD95L, CD95 ligand; LDL, lowdensity lipoprotein; EP2 receptor, prostaglandin E2 receptor 2; PPAR-,
peroxisome proliferator-activated receptor ; Iba1, ionized calcium-binding
adapter molecule 1.

MICROGLIA VERSUS MACROPHAGES:

DIFFERENCES AND SIMILARITIES
Microglia are comprised into the myeloid-monocytic compartment on the basis of markers and the immune-effector functions they can display (see above). However, this leaves a lot of
space for interpretation, especially when trying to distinguish
parenchymal microglia from infiltrating macrophages associated with a particular neuropathological lesion. Moreover,
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Journal of Leukocyte Biology Volume 85, March 2009

perivascular macrophages within the CNS are being replenished continuously by peripheral blood monocytes or bone
marrow-derived cells [146]. The story is complicated further by
the fact that infiltrating monocytes/macrophages can acquire a
phenotype in the CNS that is, at least morphologically, indistinguishable from the microglial appearance [147]. Even more,
activated amoeboid microglia, depending on the stimulus, can
exert virtually all macrophage-associated immune functions.
Nevertheless, a clear-cut distinction between these populations
is indispensable to identify initiators and/or contributors within
CNS lesions of different etiology.
The first attempt for such a division was made by flow
cytometry. In this study, microglia were differed from monocytes and macrophages based on differences in CD11b/CD45
expression: Microglia are characterized as CD11b/CD45low,
and monocytes/macrophages are characterized as CD11b/
CD45high [148]. In another study, which ex vivo-phenotyped
human microglia, these cells were also designated as CD11b/
CD45low [149]. In addition, Ulvestad and colleagues [105]
described that microglia do not express typical macrophage
markers, such as NSE, calprotectin, lysozyme, and CD14;
however, others demonstrated a low-to-moderate level of expression for these proteins [87, 147]. A multiple label definition was supplied by Dick and co-workers [150], identifying
resident microglia as CD45low:CD4:CD11b:CD11chigh:MHCII:CD26:CD14 and activated microglia as CD45low/medium:
CD4low:CD11b: CD11chigh:MHCII/:CD26:CD14 . A recent report reviews extensively the distinction between activated microglia and macrophages. In their conclusion, the
authors describe that the proliferation capacity of microglia,
the expression of laminin, CD11chigh, and CD45low, and the
absence of CD14 and peroxidase activity are specific for the
microglial phenotype, whereas macrophages display the opposite for these characteristics [147]. In addition, the authors
suggest that microglia are best distinguished from macrophages
by their spiky appearance, as demonstrated by scanning electron microscopy, and macrophages have a ruffled surface
(Rose-like aspect). Unfortunately, this method has its technical
limitations and cannot be unified in many experiments and/or
laboratories.

MICROGLIAL FUNCTION
Phagocytosis and antigen presentation
Attracted by endogenous and exogenous chemotactic factors,
microglial cells have the capacity to migrate toward a site of
injury or infection in the CNS [151, 152]. For this, they display
up-regulated expression of receptors to sense signals associated with tissue damage or inflammation and secrete proteases,
autocrine, and/or paracrine mediators, allowing them to move
through CNS tissue [90]. An orchestrated process of microglia
migration combined with proliferation allows microglia to reach
sufficient cell numbers at the site of inflammation or tissue
damage [153].
As first line of defense guardians, a typical feature of the
fully activated/capacitated microglial cell is to perform phagocytosis. As a result of the highly specialized CNS environment,
http://www.jleukbio.org

this process has to be controlled tightly, thereby provoking the

least possible collateral damage. During CNS development,
regressive phenomena accompanied by naturally occurring cell
death are common. Microglia are known to function as transitory phagocytes responsible for clearing apoptotic neuronal cell
bodies during CNS ontogeny [75]. In EAE, it has been shown
clearly that microglia are responsible for phagocytosis of infiltrated T cells that underwent apoptosis [154]. At least in vitro,
this phagocytic process involves a scavenger receptor CD36
and lectin-, integrin-, and/or phosphatidyl-mediated recognition [155, 156]. Several cytokinesamong them, GM-CSF,
M-CSF, TNF-, GF-, IL-4, IFN-, and IFN- [156 159]
are able to influence the phagocytotic properties of microglia,
but also, exogenous stimuli, such as morphine, PMA, and LPS,
can modulate uptake and processing of antigens and cellular
debris [156, 160]. The importance of phagocytosis in several
neuropathologies will be discussed in detail below (see Microglia and CNS diseases).
In the normal CNS, APC are confined mainly to dendritic
cells (DC) and macrophages of the meninges, choroid plexuses,
and perivascular spaces [129]. MHC class I and class II
molecules are constituvely expressed by microglia, although at
low levels [114, 115, 161], as they are actively down-regulated
by the immune-quiescent microenvironment of the CNS [162].
However, upon infectious neuropathology or autoimmune disease, but to a much lesser degree in trauma, ischemic, or
neurodegenerative conditions, these molecules are up-regulated readily, which convert microglia into APC comparable
with DC, the so-called experts in antigen processing and
presentation inside as well as outside the CNS. Currently, there
is accumulating evidence that under these conditions, microglia up-regulate MHC expression together with the costimulatory molecules CD11a, CD54, CD58, CD80, and CD86, all
essential for optimal APC function and T cell stimulation [163,
164]. However, to avoid excessive inflammation in the CNS, as
this reaction cannot take the same proportions as in peripheral
tissues without causing massive bystander damage, the activation of counter-regulatory mechanisms is indispensable. Indeed, for this purpose, microglia are capable of expressing Fas
ligand (FasL; CD95L) in vitro [165] and in EAE lesions [166],
implicating their role in the Fas (CD95)-FasL-mediated T cell
apoptosis. Moreover, microglia themselves can express Fas
[166], indicating that microgliosis in neuropathology can be
self-limiting by eliminating not only infiltrating, inflammatory
cells but also the excess of microglia by apoptosis.

Radical production
Depending on the species, microglia can produce different
types of free radicals. Porcine and human microglia mainly
produce superoxide radicals upon stimulation, and murine
microglia mainly produce NO [167]. Several stimulants promote NO production in rodent microglia, e.g., cytokines, A,
thrombin, zymosan, chromogranin, prions, HIV-associated Tat,
and ATP [142, 168]. On the other hand, A, IFN-, and PMA
are able to induce superoxide formation [168]. NO and superoxide production by microglia can also be modulated by proand anti-inflammatory cytokines and/or growth factors [87,
169, 170]. In addition, phagocytosis itself serves as an inducer
of free radical production, for which these reactive oxygen

TABLE 2.

Microglia and Chemokines

Chemokines

Chemokine receptors

References

CXCL1/Gro-/KC
CXCL2/Gro-
CXCL3/Gro-
MIP-2
CXCL4
CXCL8/IL-8
CXCL9/MIG
CCXCL10/IP-10
CXCL12/SDF-1
CCL1/I-309
CCL2/MCP-1
CCL3/MIP-1
CCL4/MIP-1
CCL5/RANTES
CCL19/MIP-3
CCL22/MDC
CX3CL1/Fractalkine

CXCR2
CXCR2
CXCR2
CXCR2
CXCR3
CXCR1/CXCR2
CXCR3
CXCR3
CXCR4
CCR8
CCR2
CCR1/CCR5
CCR5
CCR1/CCR3/CCR5

[172]
[173]
[173]
[152]
[174]
[173,175]
[175,176]
[175,176]
[177]
[178,179]
[180]
[181]
[181]
[182,183]
[184]
[185]
[186]

CX3CR1

Gro-, Growth-regulated oncogene-; KC, Keratinocyte-derived chemokine;

MIG, monokine induced by IFN-; IP-10, IFN-inducible protein 10; SDF-1,
stromal cell-derived factor-1; MDC, macrophage-derived chemokine.

species (ROS) are supporting the degradation of the ingested

antigens and cellular debris.

Production of immunoregulatory molecules

Chemokines

Especially during their activated state, microglia can express and

secrete a broad spectrum of chemokines [171] (Table 2). These
are small peptides (8 14 kDa) playing a crucial role in cellular
migration and intercellular communication in normal tissues, including brain, but even more during inflammation. On the basis of
their structural characteristics, according to the sequence motif of
conserved N-terminal cysteine residues, they can be divided into
four families: CXC, CC, XC, and CXC3 [187]. Chemokines act
through signaling their cognate chemokine receptors [187] and
can be up-regulated in microglia in vivo during normal CNS
development (CXCL1 [172]) but also under neuropathological
conditions such as multiple sclerosis (MS; CCL3/MIP-1 [188]),
ischemia (CXCL8/IL-8 [173]), Alzheimer disease (AD; CCL2/
MCP-1 [189]), and different bacterial (CCL5/RANTES [190]) or
viral infections (CCL4/MIP-1 [181]). In addition, microglial proliferation/survival, migration, cytoskeleton reorganization, and cytokine production can be affected by chemokines acting through
several receptors found on microglia (Table 2). Also, during microglia colonization of the CNS, a major role has been attributed
to the expression of, e.g., CCL2/MCP-1 and CCL5/RANTES in
nervous parenchyma [66].
Cytokines

Another group of important signaling molecules for microglia

includes a large number of cytokines (Table 3). These small
proteins are usually expressed/secreted by microglia under inflammatory conditions. Important exogenous factors capable of
cytokine induction in microglia are viral envelope proteins, bacterial cell wall components [LPS, leukotriene A (LTA)], bacterial
DNA, and prions [214]. Endogenous inducers are inflammatory
mediators, such as platelet-activating factor (PAF), lipids, serum
Tambuyzer et al. An introduction to microglia

357

TABLE 3.

Microglia and Cytokines

Cytokine

Cytokine receptors

References

IL-1/
IL-1ra

IL-1R
IL-1R
IL-2R
IL-3R
IL-4R
IL-5R
IL-6R
IL-8R
IL-10R
IL-12R
IL-13R
IL-15R

[191]
[192]
[193]
[194,195]
[196,197]
[198,199]
[198]
[198]
[198]
[198]
[198,200]
[198]
[201]
[202]
[203,204]
[205,206]
[207]
[208]
[209]
[210,211]
[212]
[213]

IL-3
IL-4
IL-5
IL-6
IL-8
IL-10
IL-12
IL-13
IL-15
IL-16
IL-17
IL-18
IL-23
IL-27
TNF-
TGF-
IFN-
GM-CSF
M-CSF

IL-18R
IL-23R
TNFR
TGFR
IFN-R
GM-CSFR
M-CSFR

IL-1ra, IL-1 receptor antagonist.

proteins, or complement factors, but also, disturbed ATP and [K]

levels can cause microglial activation [133].
IL-1 and TNF- are two proinflammatory cytokines with
diverse influences and many common functions, which can be
produced by microglia [215] and can also serve as autocrine
activators [216, 217]. These cytokines are mainly involved
during initiation of neuroinflammation by their ability to induce
expression of the adhesion molecules and chemokines necessary for attraction of leukocytes [218, 219]. In addition, these
processes can be aggravated by IFN-, mainly produced by
CD4, CD8 T cells, and NK cells but also by macrophages/
microglia upon an IL-12- and IL-18-mediated stimulation,
thriving the immune response toward a cytotoxic response
(mostly mediated by induction of a Th1 response [129]). To this
end, microglia themselves can secrete IL-12 and IL-18,
thereby providing a self-stimulating loop and activation of NK
and Th1 cells [220, 221]. In turn, microglia are activated and
primed for phagocytosis and antigen presentation by IFN-
through expression of all receptors necessary for an adequate T
cell stimulation [133].
IL-2, mainly involved in T cell expansion, and IL-15, a
cytokine serving as a partial substitute for IL-2, can influence
microglial behavior [193]. Both cytokines share receptors subunits, which explains their overlapping effects. IL-2 stimulates
microglia by increasing their survival and NO production
[133]. IL-15, which can also be produced by microglia themselves, selectively activates NK and CD8 cells but has an
attenuating influence on microglia [193]. IL-6 is an additional
pleiotropic cytokine produced by microglia with pro- and antiinflammatory properties responsible for promoting a cell-mediated humoral immune reaction (induction of Th2 response
[222]).
On the other hand, microglia can also produce a number of
anti-inflammatory cytokines, such as IL-10, TGF-, and IL-1ra
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Journal of Leukocyte Biology Volume 85, March 2009

[192, 223, 224], which inhibits the actions of IL-1, and IL-10
and TGF- down-regulate the expression of proinflammatory
cytokines, ROS, chemokines, and molecules associated with
phagocytosis on microglia [225227]. In addition, Th2 cells
that are able to produce IL-4 can counteract microglial activation [227]. It has been demonstrated that microglia can
produce IL-4 and IL-13 themselves; however, autocrine stimulation seems to induce cell death [228]. In this context, cell
loss of activated microglia might serve as an ultimate tool to
prevent overactivation and increase neuronal survival [200].
Growth factors

GM-CSF, IL-3, and M-CSF are important inducers for microglial proliferation [82, 133]. Moreover, GM-CSF stimulates free
radical production by microglia [87] and enhances microglial
phagocytosis [157]. In addition to their proliferation-stimulatory effect, IL-3 induces MHC class II up-regulation [229], and
M-CSF promotes phagocytosis by microglia [158].
Prostanoids

PGs and thromboxanes play an important role in immune

reactions. These prostanoids are potent down-regulators of
microglial activation through the EP2 receptor and PPAR-
(Table 1) by inhibiting the production of proinflammatory cytokines (IL-1 [230]; IL-12/23/27 [207]; IL-18 [231]) and NO
and the expression of MHC class II and costimulatory molecules [232]. Th1 cells, whose function is also down-regulated
by PGE2, can supply a feedback to microglia for bursting PGE2
production [233]. To date, it has been shown that microglia, in
vivo and/or in vitro, can express EP1, -2, -3, and -4 [230, 234,
235], but the function of these receptors (except for EP2) in
microglial cell biology and their immunologic profile have yet
to be elucidated.

TLR-mediated regulation of microglia

TLRs are a class of receptors that enable immune cells to
recognize pathogen-associated molecular patterns (e.g., LPS,
LTA, unmethylated CpG, dsRNA/DNA, peptidoglycan, and
others) [236] but also, endogenous activators such as heat
shock proteins, extracellular matrix molecules, and necrotic
cells [237]. At least in mice, it has been demonstrated that
primary cultured microglia can express TLR19 (of 13 known
mouse TLRs) [117], and human microglia express TLR1 8 (of
11 known human TLRs) [42]. Ligation of these receptors leads
to microglial expression of proinflammatory cytokines and chemokines [117, 238, 239], thereby evoking an innate immune
response against viral and/or bacterial pathogens. For example,
TLR2 signaling is indispensable for microglial responses
against group B Streptococcus, mainly mediated by NO production [240]. The same receptor also evokes the production of
immune molecules following in vitro infection of microglia with
HSV-1 [241]. Moreover, TLR signaling serves as an inducer of
APC functions by up-regulating MHC II and costimulatory
molecules [117]. These TLRs also appear to be implicated in
microglia homeostasis and in different neuropathologies: LPS
stimulation of microglia (TLR4-mediated) results in increased apoptosis mediated by autocrine/paracrine production
of IFN- by microglia themselves [242]; in a mouse model for
AD, TLR4 deficiency impairs microglia to clear A adequately
http://www.jleukbio.org

in vitro and in vivo [243]; TLR2 can become up-regulated in

microglia following cerebral ischemic injury, thereby mediating postischemic damage, at least in mouse CNS [244]. Not
surprisingly, TLR-stimulated microglia can be toxic to oligodendrocytes as well as neurons in their vicinity, a consequence
reported in vitro and in vivo [245247]. Possibly, this bystander damage can be attenuated by PGs, such as 15d-PGJ2.
The latter down-regulates microglial TLR2 expression and
subsequent downstream effects [248] and thereby inhibits
TLR-evoked cytokine (IL-12, TNF-) and NO production by
microglia [249]. PGs may thus provide a counter-regulatory
feedback for TLR stimulation of microglia when aiming to
balance between a beneficial versus detrimental role for these
cells during neuroinflammation.

Giant cell formation

In some neuropathology conditions, e.g., AIDS-associated dementia and tuberculous meningitis, microglia acquire the
highly activated phenotype of a multinucleated giant cell [250,
251], and these giant cells are formed by fusion of multiple
microglia, thereby producing large amounts of free radicals and
proteolytical enzymes. Although the mechanism of giant cell
formation still needs to be clarified further, at least this process
can be affected by several cytokines and/or growth factors.
IL-3, IL-4, IL-13, and GM-CSF promote the fusion of rodent
microglia [252, 253]. In contrast, fusion of porcine microglia is
inhibited by GM-CSF [112] but stimulated by TNF- [254].

Neuroimmune regulatory proteins

As mentioned above, microglia are kept in a quiescent state
mainly through CD200 CD200R interaction, an inhibitory
mechanism demonstrated to be impaired in EAE/MS [79, 255],
experimental autoimmune uveoretinitis [256], and ischemic
brain injury [257]. At least in part, this effect is mediated by
IL-4, as demonstrated by in vivo and in vitro studies [258]. In
addition, CD47, through interaction with signal regulatory protein , inhibits phagocytosis by macrophages [259] and is
important in MS physiopathology [255]. Also, CD22, expressed
and secreted by neurons, down-regulates production of proinflammatory cytokines by microglia through interaction with the
CD45 surface receptor [260]. Scavenger receptors are another
important group of surface molecules affecting microglial cell
biology and function. As mentioned before, microglia are susceptible for Fas/FasL-mediated elimination, possibly by neighboring microglia with a regulatory function. Griffiths and colleagues [261] described neuroimmune regulation of microglia
extensively.

MICROGLIA AND CNS DISEASES

AD
AD is a neurodegenerative disorder characterized by three
major pathological findings within the CNS: neuritic plaques
(NPs) constituted mainly of -amyloid aggregates, a degradation product of the amyloid precursor protein (APP); neurofibrillary tangles (NFTs) formed by hyperphosphorylated microtubule-associated protein ; and neuroinflammation induced by

(micro)gliosis. The number of NFTs correlates more with the

severity of AD than the number of NPs [262]. However, the
best correlate for the cognitive decline associated with AD is
the synaptic/neuronal loss induced in part by microglia-mediated inflammatory responses [262]. At least in the mouse
model, microglial toxicity in the AD brain appears almost
directly after plaque formation and leads to further damage to
the surrounding neuritic population [263]. Microglia in the
AD-affected brain show an up-regulation of MHC class I/II,
FcR, integrin, vitronectin, and scavenger receptors [264, 265].
Important proinflammatory cytokines, among them IL-1,
TNF-, and IL-6, are secreted by microglia and astrocytes
[266, 267], further stimulating microglia to produce neurotoxins, specifically in lesioned CNS areas. These toxins comprise
ROS, lipophilic amines, quinolinic acid, excitatory amino acids, and PAF and may themselves amplify glial activation
responses [262, 268].
Furthermore, -amyloid or APP can activate microglia, resulting in neuronal death [268, 269]. This again pinpoints
microglia to be an important source for secreting neurotoxic
substances. In this context, scavenger receptors and heparan
sulfate, a docking site for cell binding used by apolipoprotein
E (apoE), have been suggested for plaque recognition through
binding of -amyloid [268]. In experimental set-up, microglia
have been shown to produce proinflammatory cytokines, chemokines, and ROS upon stimulation with the -amyloid peptide (A1-42) [266, 270]. Moreover, proinflammatory cytokines
can up-regulate APP expression, thus providing extra building
stones for -amyloid production and sustaining a vicious cycle
of inflammation [271]. In addition, chromogranins, a group of
acidic glycoproteins, are found in NPs and dystrophic neurons
[272] and can induce microglia to produce neurotoxic products
[273].
All complement factors needed to form the membrane
attack complex (MAC) have been demonstrated to be present
in the brain of AD patients. -Amyloid can aggregate with the
C1q protein, which in turn strongly binds the C1q receptor on
microglia, leading to their activation [274, 275]. In addition,
the activation of complement by -amyloid aggregates is potentiated by apoE [276]. In this way, the classical complement
pathway is initiated without the presence of Igs. This phenomenon of microvascular -amyloid deposition, a common pathological feature of AD, evokes an up-regulation of three complement proteins (C1q, C3, and C4) by surrounding microglia
[277]. Eventually, the MAC will not distinguish friend from foe,
leading to important neuronal loss. Although many complement inhibitors are also up-regulated in AD, these molecules
probably cannot prevent the overwhelming bystander damage
[278]. In conclusion, the inflammatory response in AD seems to
be secondary to the formation of NPs and NFTs. The aggregates
serve as stable irritants for microglia, leading to disease progression by a self-sustained inflammatory cycle [279]. This
hypothesis is supported further by epidemiological and experimental animal studies using nonsteroidal, anti-inflammatory
drugs, which demonstrate delayed disease onset and/or progression [280]. However, initially, microglial reaction can also
be beneficial when aiming to clear -amyloid by phagocytosis
and secretion of proteases, e.g., neprilysin and insulin-degrading enzyme [281].
Tambuyzer et al. An introduction to microglia

359

MS
MS is a chronic, demyelinating, inflammatory disease of the
CNS with an important autoimmune component [282]. Without
clear understanding of the events initiating MS, the pathogenesis of the lesions associated with this disease can be divided
into distinct patterns. This distinction is made based on deposition of Igs and complement, location of demyelination and
remyelination, and type of oligodendroglial cell death, apoptosis or necrosis [283]. However, all patterns and lesions have
one common feature: Infiltration and activation of mostly myelin-specific T lymphocytes and myeloid cells are always found
within areas of active demyelination. The contribution of microglia in MS pathogenesis remains controversial. Although
microglia are able to capture, process, and present myelin as
an antigen [284], research has not pinpointed these cells
univocally as the main initiators of MS nor to its experimental
counterpart EAE [285]. In addition, infiltration of macrophages
and/or DC seems required, and microglia become activated
simultaneously or subsequently as a result of immune crosstalk
with infiltrating immune cells via chemokine and IL signaling
[285288]. In addition, a complex interplay with complement
factors and/or Igs, myelin itself is able to stimulate microglia,
further aggravating a microglial response by stimulating their
production of neuro- and glio-toxins.
IFN- therapy, today the most important clinical strategy to
delay MS [289], may anticipate microglial toxicity against
neurons, as shown by in vitro research [290]. On the other
hand, microglial responses are regulated strictly by the immunosuppressive CNS microenvironment and might induce Fas/
FasL-mediated apoptosis of the infiltrating, myelin-specific T
cells [291]. In addition, it has been reported recently that
microglia can suppress T cells through the programmed death
receptor-1 in the EAE mouse model, thereby down-regulating
Th1 cells rather than Th17 cells [292]. Microglia, at least in
EAE, can be skewed by IL-4 to display a more beneficial
activation state, devoid of neurotoxic NO production [293]. On
the other hand, microglia associated with MS lesions, do show
an elevated expression of myeloperoxidase and thus, potentially contribute to oxidative stress leading to the demyelination [294]. In contrast, CCR5 expressed on microglia has been
reported to be associated with remyelination in MS lesions
[295]. Although microglia themselves are not designated as
initiators of MS/EAE, new, potential therapeutic agents, which
act specifically by down-regulating microglial activation, do
contribute to a better disease outcome in EAE [296]. Altogether, these reports point out a complicated role for microglia
in MS/EAE: perhaps detrimental in the initial phase, responsive to the proinflammatory environment created by infiltrating
T cells, but at a later stage, beneficial by clearing CNS parenchyma of T cells and myelin to control further damage and to
rescue oligodendrocytes as well as neurons.

Parkinsons disease (PD)

PD is a neurodegenerative disease typed by degeneration of
dopaminergic neurons in the SN pars compacta and the loss of
their projecting fibers in the striatum [297]. Highly elevated
levels of IFN-, TNF-, and IL-1 are detected in the SN of
PD patients. Microglia can be a source for these cytokines
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Journal of Leukocyte Biology Volume 85, March 2009

(Table 3), and dopaminergic neuronal degeneration caused by

activated microglia has been reported. However, especially in
the case of IFN-, in situ expression of this cytokine remains
to be demonstrated. In fact, microglial activation seems an
early event in PD neuropathogenesis, as shown by a gradual
phagocytosis of nigral dopamine neurons by large, ramified
microglia [298]. The activation of microglia in PD is mediated
by at least three proteins released by damaged neurons: matrix
metalloproteinase 3 (MMP-3), -synuclein, and neuromelanin.
MMP-3 secretion by dopaminergic neurons causes an augmented microglial production of superoxide and TNF- [298,
299]. -Synuclein induces ROS production and secretion by
microglia [300]. Next to toxic molecules, microglia also secrete
neuroregulatory proteins upon -synuclein stimulation, suggesting a dual response of microglia in PD [301], although the
latter is not able to prevent PD. Neuromelanin, a dark, insoluble macromolecule that serves as an antioxidant within neurons and has neuroprotective properties through dopamine and
metal binding, exerts chemotactic effects and induces microglial production of TNF-, IL-6, and NO [302].

Creutzfeldt-Jacob disease (CJD)

CJD belongs to a class of transmissible spongiform encephalopathies, characterized by the deposition of protease-resistant
isoforms [disease-specific prion protein (PrPSc)] of PrP [303].
Neuronal loss in prion diseases is mostly attributable to programmed cell death [304]. In vivo and in vitro models suggest
that microglial activation precedes this apoptosis and is indispensable for the neurotoxicity of PrPSc [305]. Astrocytes and
neurons recruit microglia early in prion disease through secretion of RANTES and MIP-1. This process is CCR5-mediated,
as shown by a decrease in microglial attraction by a specific
CCR5 antagonist, TAK-779 [306]. Apparently, microglia-secreted factors (e.g., NO) consequently induce neuronal apoptosis [307]. In addition, PrPSc itself disables microglia to perform
efficient phagocytosis of PrPSc [308]. Despite the fact that MHC
II expression is readily up-regulated in prion disease, which is
associated with the active function of microglia, CJD is classified with diseases of chronic neuroinflammation without overt
T cell involvement [309].

Brain tumors
Infiltrative astrocytoma are characterized by the occurrence of
large numbers of microglia/macrophages. One-third of all cells
in glioma biopsies expresses microglial/macrophage markers
[310]. Activated amoeboid microglia are mostly associated with
high-grade tumors such as glioblastoma [311], and ramified
microglia are found more in gemistocytic astrocytoma [312].
Microglia can be attracted to the tumor site by chemokines
(e.g., MCP-1) and growth factors (e.g., CSF-1 and G-CSF),
locally produced by tumorigenic astrocytes [313315]. In addition, these growth factors contribute to proliferation of microglia within astrocytoma.
Despite their presence, immune function of microglia in
astrocytoma is likely to be suppressed. Although they are
relatively abundant, the number of MHC class II expressing
microglia is reduced in high-grade astrocytic gliomas [316].
Indeed, it has been shown that tumor-associated microglia fail
http://www.jleukbio.org

to express MHC II upon stimulation [317]. Glioma culture

supernatants can suppress IFN--induced MHC-II expression
on microglial cells, thereby impairing their ability for proper
antigen presentation and thus, preventing the induction of an
efficient anti-tumor immune response [318]. Brain tumors can
also produce anti-inflammatory cytokines, e.g., IL-6 and
TGF-, which potently down-regulate microglial immune-effector functions [319]. This immunosuppressive environment is
enhanced further via IL-10 production by microglia themselves
[320]. The latter cytokine is up-regulated in microglia by a
tumor-associated, lower expression of upstream stimulating
factor-1 [321]. Even more, IL-6 and IL-10 have been associated with tumor growth and infiltration [322, 323]. To this
extent, microglia secrete endothelial growth factor (EGF), vascular EGF (VEGF), and hepatocyte growth factor (HGF) in
brain tumors, all of which are associated with cell growth,
metastasis, and angiogenesis [324].

HIV-associated neuropathology
Entry of HIV in the CNS is actively mediated via microglia or
perivascular macrophage infection, and neurons and other glia
are not likely to be active carriers. How HIV might infect
microglia remains elusive, as they do not express detectable
levels of the CD4 receptor. Antibody-mediated uptake of virus
via FcRs expressed at the microglial cell surface has been
suggested as an alternative mechanism for HIV infection [325],
although the chemokine receptor CCR5, also expressed by
microglia, is currently considered as the most important gate
for viral entrance [326]. However, others question microglia as
a source of viral reproduction and ascribe sustained infection
of the CNS to perivascular macrophages, which are replenished
continuously by HIV-infected peripheral blood monocytes
[327].
HIV-associated dementia (HAD) has been correlated with
activation of microglia and macrophages in brain tissue. The
latter is the major cause of neuronal damage or loss within the
CNS following HIV infection [328]. A typical hallmark of HAD
is multinucleated giant cell (MNGC) formation. These cells
originate from the fusion of microglia and/or macrophages to
become a highly activated cell. MNGC produce excessive
amounts of ROS, enzymes, but also viral proteins [329, 330].
Among the latter, viral HIV-I Tat, gp120, and Vpr can act as
direct neurotoxic substances or add to the bystander damage by
further stimulating inflammation [331]. This innate and viral
neurotoxic cocktail, together with the production of ILs and
chemokines, which attract even more inflammatory cells, is
suggested to account for the major neuronal loss associated
with HAD.

Miscellaneous neuropathology-associated stimuli

for microglial activation
The main cause of encephalitis or meningitis (for a review, see
ref. [332]) includes viruses such as HIV, varicella zoster virus,
human herpes viruses 6 and 7, EBV, cytomegalovirus, enteroviruses, adenoviruses, and influenza virus A/B. However, bacteria, rickettsiae, fungi, and protozoae are also potential intruders of the CNS. Major consequences of infection might
include edema, hemorrhage, brain abscess, demyelination,

neuronophagy, and necrosis, dependent on the source of infectious material. In response to all of these infections, microglia
become activated and might produce a number of proinflammatory cytokines and chemokines, up-regulate phagocytosis
and ROS production, and even serve as a reservoir.
Microglia are the first immune cells to sense damage to the
CNS caused by a traumatic injury, upon which they react by
increased expression of excitatory amino acid transporters
(EAAT). Injury-associated cortical cell loss and concomitant
increase in excitatory amino acids add to secondary brain
damage, whereas microglia perhaps try to compensate for these
events with a potential neuroprotective response [333]. Another type of cerebral injury occurs during ischemia or stroke.
Hereby, microglia are stimulated to produce a broad spectrum
of immune molecules. Synthesis and release of complement
factor C1q by microglia are highly increased upon ischemic
injury [334]. Neuronal secretion of fractalkine and MCP-1 is
up-regulated in response to ischemia, whereas microglia produce increased levels of MIP-1 and MIP-2 [335337]. Simultaneously, the expression of chemokine receptors CCR5 and
the fractalkine receptor CX3CR1 on a microglial surface is
increased [335, 336]. Following ischemia, microglia display
elevated production of potentially neurotoxic IL-1 and TNF-
and TNFR1/2 [338, 339]. IL-6 is produced by microglia after
ischemia, although this expression returns to basal level a few
hours post-insult [340]. In ischemic events, this cytokine and
its close relative ciliary neurotrophic factor decrease neuronal
death [341, 342]. Platelet-derived growth factor and glial cell
line-derived neurotrophic factor are two neurotrophic factors
produced by post-ischemic microglia able to promote neuronal
survival [343, 344]. The anti-inflammatory cytokine TGF-,
secreted by microglia after ischemia, can also promote neuronal survival directly or have an autocrine/paracrine influence
in which microglial overactivation and secondary neuronal
damage are prevented [345]. Analogously to traumatic brain
injury, microglia can up-regulate EAAT to scavenge glutamate
and may reduce its neurotoxic potential [345]. However, it
should be mentioned that microglia (in contrast with astrocytes), despite their expression of EAAT, are not capable of
reducing the neurotoxicity of glutamate in vitro [346]. In conclusion, microglia also have been demonstrated to produce
several neurotrophins (NTs; e.g., brain-derived neurotrophic
factor, nerve growth factor, NT-3/4/5) and/or growth factors
(e.g., insulin-like growth factor-1, basic fibroblast growth factor, HGF, VEGF, EGF) potentially beneficial to neuronal survival [145].

EXPERIMENTAL MODELS FOR MICROGLIA

RESEARCH IN VITRO AND IN VIVO
Throughout literature, in vitro culture models for microglia are
usually based on the same methodology, although variations
may exist to investigate different aspects of microglial cell
biology or to simulate different neuropathological disease conditions. The short overview below briefly describes current
possibilities for studying microglia in vitro.
Microglia can be obtained from mixed cortical glial cultures
originating from embryonic/fetal, adult, or diseased CNS of
Tambuyzer et al. An introduction to microglia

361

TABLE 4.

Models for Microglia in Neuropathology

Disease
AD
MS
PD
CJD

Model

Transgenic mice
EAE
MPTP mice
Scrapie, BSE (animal disease
anologues)
Brain tumors
Experimental gliomas
HAD
SIV (animal analogue)
Brain abcess
Experimentally induced
abcess
Brain trauma
Stab wound, closed head
injury
Stroke
Experimental ischemia
Peripheral nerve injury - Facial nerve axotomy
(retrograde degeneration)
- Optic nerve axotomy
(Wallerian, anterograde
degeneration)

Reference
[352]
[353]
[354]
[355]
[356]
[357]
[358]
[359, 360]
[361]
[362, 363]
[364, 365]

MPTP, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; BSE, bovine spongiform encephalopathy.

different species (rat [35]; swine [160]; human [266]; goldfish

[347]; quail [348]). These cultures are started as a monolayer,
mainly consisting of astrocytes with few contaminating fibroblasts, endothelial cells, and oligodendrocytes. After 2 weeks,
confluence is reached, and loosely adherent microglia proliferate actively on top of the astrocyte feeder layer. Their proliferation is stimulated mainly by growth factors (e.g., GM-CSF,
CSF-1), produced by this feeder layer, and microglia can be
recovered by shaking the culture plate. The main challenge of
this culture model is to reinduce the ramified morphology of
microglia, which is usually lost by activation during tissue
preparation. At least in vitro, this can be established by adding
growth factors (e.g., GM-CSF [87]) or through direct contact
with an epithelial monolayer [82]. Although most experiments
are performed using primary microglia cultures, a few microglial cell lines have been described. Although BV-2 and N9
mouse microglial cell lines [349] are well described in literature, laboratory-specific microglia cell lines also exist [350]. In
addition, an important organotypic model for microglia research is the retina [351]. This can be removed without contaminating tissue and allows surveyable analysis of ramified
microglia in situ. The organotypic brain slice preparation [36]
has been proposed as an alternative for whole mount culture.
However, like mostly observed during microglial monoculture,
microglia might acquire an activated state as a result of manipulation and culture conditions.
Throughout literature, several animal models for microglia
research have been well characterized and are discussed partially within this review. Table 4 provides an overview of
several in vivo research models, which might serve as a basis
for in vivo microglia research in neuropathology.

CONCLUDING REMARKS
As a result of their versatile phenotype and the lack of appropriate biomarkers, microglia were often misidentified in the
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past. However, nowadays, microglia can be distinguished

clearly from other CNS populations using an extensive microglia-defining set of molecules together with their highly specialized immunologic function. Moreover, it is clear now that
microglia are involved in virtually all neuropathological processes. Within inflamed CNS (e.g., infections, autoimmune
diseases), they can cause primary damage to neurons, and in
neurodegenerative disease (e.g., AD, PD), microglia become
highly reactive to dying neuronal cells and provoke secondary
neurotoxicity. Therefore, the research aim for neuroscientists
and immunologists has now shifted toward investigating the
balance of a beneficial versus a detrimental phenotype of
microglia in the CNS tissue. On the one hand, they can display
good guy characteristics by producing neurotrophic growth
factors and clear endogenous and exogenous neurotoxins (e.g.,
excitatory amino acids) and eliminate unwanted inflammatory
cells (e.g., autoreactive T cells) from the nervous parenchyma.
On the other hand, they can aggravate CNS injury by production of neuro- and/or gliotoxins (e.g., ROS, NO, enzymes) and
thus cause a sustained, vicious cycle of microglial activation
with subsequent secondary neural damage. However, it should
be kept in mind that microglial actions are controlled by
several neuroimmune regulatory mechanisms and are adapted
to the CNS environment. Indeed, compared with its peripheral
counterpart (i.e., macrophages), microglia seem to be restricted
and/or limited in their immune functions, although their capacity to proliferate in situ might increase their power instantaneously. Perhaps these characteristics allow them to screen
and protect the CNS in a gentle, neuron-friendly manner.

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