Key Event Title
|Level of Biological Organization|
Key Event Components
|cell activation involved in immune response||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Oxidative stress and Developmental Neurotoxicity||KeyEvent|
|Macaca fascicularis||Macaca fascicularis||NCBI|
|All life stages|
Key Event Description
Tissue resident cell activation is considered as a hallmark of inflammation irrespective of the tissue type. Strategically placed cells within tissues respond to noxious stimuli, thus regulating the recruitment of neutrophil and the initiation and resolution of inflammation (Kim and Luster, 2015). Examples for these cells are resident immune cells, parenchymal cells, vascular cells, stromal cells, or smooth muscle cells. These cells may be specific for a certain tissue, but they have a common tissue-independent role.
Under healthy conditions there is a homeostatic state, characterized as a generally quiescent cellular milieu. Various danger signals or alarmins that are involved in induction of inflammation like pathogen-associated molecular pattern molecules (PAMPs) and damage-associated molecular pattern molecules (DAMPs) activate these resident cells in affected tissues.
Examples of well-characterized DAMPs (danger signals or alarmins) (Saïd-Sadier and Ojcius, 2012)
Outcome of receptor ligation
PI, P2X and P2Y receptors (ATP, ADP); Al, A2A, A2B and A3 receptors (adenosine)
Dendritic cell (DC) maturation, chemotaxis, secretion of cytokines (IL-1β, IL-18), inflammation
Extracellular heat shock
CD14, CD91, scavenger
DC maturation, cytokine induction, DC, migration to lymph nodes
RAGE, TLR2, TLR4
Chemotaxis, cytokine induction, DC activation, neutrophil recruitment, inflammation, activation of immune cells
Uric acid crystals
CD14, TLR2, TLR4
DC activation, cytokine induction, neutrophil recruitment, gout induction
Intracellular redox-sensitive proteins
Cell death, release of endogenous DAMPs, inflammation
Neutrophil recruitment, chemotaxis
S100 proteins or
Neutrophil recruitment, chemotaxis, cytokine secretion, apoptosis
TLR2, TLR4, CD44
DC maturation, cytokine production, adjuvant activity
Activation refers to a phenotypic modification of the resident cells that includes alterations in their secretions, activation of biosynthetic pathways, production of pro-inflammatory proteins and lipids, and morphological changes. While these represent a pleiotropic range of responses that can vary with the tissue, there are a number of common markers or signs of activation that are measurable.
Examples of Common markers are
These described commonalities allow the use of this KE as a hub KE in the AOP network. However, despite the similarities in the inflammatory process, the type of reactive cells and the molecules triggering their reactivity may be tissue-specific. Therefore, for practical reasons, a tissue specific description of the reactive cells and of the triggering factors is necessary in order to specify in a tissue-specific manner, which cell should be considered and what should be measured.
The most easily detectable feature of brain inflammation or neuroinflammation is activation of microglial cells and astrocytes. It is evidenced by changes in shape, increased expression of certain antigens, and accumulation and proliferation of the glial cells in affected regions (Aschner, 1998; Graeber & Streit, 1990; Monnet-Tschudi et al, 2007; Streit et al, 1999; Kraft and Harry, 2011; Claycomb et al., 2013). Upon stimulation by cytokines, chemokines or inflammogens (e.g. from pathogens or from damaged neurons), both glial cell types activate inflammatory signaling pathways, which result in increased expression and/or release of inflammatory mediators such as cytokines, eicosanoids, and metalloproteinases (Dong & Benveniste, 2001) (cf KE: pro-inflammatory mediators, increased), as well as in the production of reactive oxygen species (ROS) and nitrogen species (RNS) (Brown & Bal-Price, 2003). Different types of activation states are possible for microglia and astrocytes, resulting in pro-inflammatory or anti-inflammatory signaling, and other cellular functions (such as phagocytosis) (Streit et al., 1999; Nakajima and Kohsaka, 2004). Therefore, neuroinflammation can have both neuroprotective/neuroreparative and neurodegenerative consequences (Carson et al., 2006; Monnet-Tschudi et al, 2007; Aguzzi et al., 2013 ; Glass et al., 2010). Under normal physiological conditions, microglial cells survey the nervous system for neuronal integrity (Nimmerjahn et al, 2005) and for invading pathogens (Aloisi, 2001; Kreutzberg, 1995; Kreutzberg, 1996; Rivest, 2009). They are the first type of cell activated (first line of defense), and can subsequently induce astrocyte activation (Falsig, 2008). Two distinct states of microglial activation have been described (Gordon, 2003; Kigerl et al, 2009; Maresz et al, 2008; Mosser & Edwards, 2008; Perego et al; Ponomarev et al, 2005): The M1 state is classically triggered by interferon-gamma and/or other pro-inflammatory cytokines, and this state is characterized by increased expression of integrin alpha M (Itgam) and CD86, as well as the release of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6), and it is mostly associated with neurodegeneration. The M2 state is triggered by IL-4 and IL-13 (Maresz et al, 2008; Perego et al, 2011; Ponomarev et al, 2007) and induces the expression of mannose receptor 1 (MRC1), arginase1 (Arg 1) and Ym1/2; it is involved in repair processes. The activation of astrocytes by microglia-derived cytokines or TLR agonists resembles the microglial M1 state (Falsig 2006). Although classification of the M1/M2 polarization of microglial cells may be considered as a simplification of authentic microglial reaction states (Ransohoff, 2016), a similar polarization of reactive astrocytes has been described recently Liddlelow et al., 2017): Interleukin-1 alpha (IL-1a), TNF and subcomponent q (C1q) released by activated microglial cells induce A1-reactive astrocytes, which lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis and induce the death of neurons and oligodendrocytes.
Regulatory examples using the KE
Measurement of GFAP in brain tissue, whose increase is a marker of astrocyte reactivity, is required by the US EPA in rodent toxicity studies for fuel additives (40 CFR 79.67), but is optional for other toxicant evaluations.
How It Is Measured or Detected
Measurement targets are cell surface and intracellular markers; the specific markers may be cell and species-specific.
Available methods include cytometry, immunohistochemistry, gene expression sequencing; western blotting, ELISA, and functional assays.
Neuroinflammation, i.e. the activation of glial cells can be measured by quantification of cellular markers (most commonly), or of released mediators (less common). As multiple activation states exist for the two main cell types involved, it is necessary to measure several markers of neuroinflammation:
- Microglial activation can be detected based on the increased numbers of labeled microglia per volume element of brain tissue (due to increase of binding sites, proliferation, and immigration of cells) or on morphological changes. A specific microglial marker, used across different species, is CD11b. Alternatively various specific carbohydrate structures can be stained by lectins (e.g. IB4). Beyond that, various well-established antibodies are available to detect microglia in mouse tissue (F4/80), phagocytic microglia in rat tissue (ED1) or more generally microglia across species (Iba1). Transgenic mice are available with fluorescent proteins under the control of the CD11b promoter to easily quantify microglia without the need for specific stains.
- The most frequently used astrocyte marker is glial fibrillary acidic protein, GFAP (99% of all studies) (Eng et al., 2000). This protein is highly specific for astrocytes in the brain, and antibodies are available for immunocytochemical detection. In neuroinflamatory brain regions, the stain becomes more prominent, due to an upregulation of the protein, a shape change/proliferation of the cells, and/or better accessibility of the antibody. Various histological quantification approaches can be used. Occasionally, alternative astrocytic markers, such as vimentin of the S100beta protein, have been used for astrocyte staining (Struzynska et al., 2007). Antibodies for complement component 3 (C3), the most characteristic and highly upregulated marker of A1 neurotoxic reactive astrocytes are commercially available.
- All immunocytochemical methods can also be applied to cell culture models.
- In patients, microglial accumulation can be monitored by PET imaging, using [11C]-PK 11195 as a microglial marker (Banati et al., 2002).
- Activation of glial cells can be assessed in tissue or cell culture models also by quantification of sets of M1/M2 phenotype markers. This can for instance be done by PCR quantification, immunocytochemistry, immunoblotting.
- Itgam, CD86 expression as markers of M1 microglial phenotype
- Arg1, MRC1, as markers of M2 microglial phenotype
(for descriptions of techniques, see Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al., 2014)
Domain of Applicability
Extend to at least invertebrates
Not to plants and not to single-celled organisms
Neuroinflammation is observed in human, monkey, rat, mouse, and zebrafish, in association with neurodegeneration or following toxicant exposure. Some references (non-exhaustive list) are given below for illustration:
In human: Vennetti et al., 2006
In monkey (Macaca fascicularis): Charleston et al., 1994, 1996
In rat: Little et al., 2012; Zurich et al., 2002; Eskes et al., 2002
In mouse: Liu et al., 2012
In zebrafish: Xu et al., 2014.
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Banati, R. B. (2002). "Visualising microglial activation in vivo." Glia 40: 206-217.
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Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM. 1996. Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. NeuroToxicology 17: 127-138.
Charleston JS, Bolender RP, Mottet NK, Body RL, Vahter ME, Burbacher TM. 1994. Increases in the number of reactive glia in the visual cortex of Macaca fascicularis following subclinical long-term methyl mercury exposure. ToxicolApplPharmacol 129: 196-206.
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Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, et al. 2017. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541(7638): 481-487.
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Liu Y, Hu J, Wu J, Zhu C, Hui Y, Han Y, et al. 2012. alpha7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation. J Neuroinflammation 9: 98.
Lund S, Christensen KV, Hedtjärn M, Mortensen AL, Hagberg H, Falsig J, Hasseldam H, Schrattenholz A, Pörzgen P, Leist M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J Neuroimmunol. 2006 Nov;180(1-2):71-87.
Maresz K, Ponomarev ED, Barteneva N, Tan Y, Mann MK, Dittel BN (2008) IL-13 induces the expression of the alternative activation marker Ym1 in a subset of testicular macrophages. J Reprod Immunol 78: 140-148
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