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Differential
Response of
Microglia to
Receptor
Activation
RESTING
Multiple processes sample
surrounding area
Secrete IL-10 (Langmann 2007)
ACTIVATED
Migration
Proliferation
Phagocytosis
Secretion of cytokines/chemokines
May return to resting state after activation
Linked to neuron clearing during neural development (Block and Hong, 2005)
(Hanisch and Kettenmann, 2007)
(Neumann et al, 2008-2)
Extracellular
Peptidoglycan
TLR2
Cytoplasmic membrane
Intracellular
Dr. Porter, MICR 450 lecture, 2008
(Olsen and Miller, 2004)
Ligand
Receptor
Source
Reference
Gangliosides
TLR4
cell wall
Jou et al, 2006
ATP, ADP
Purinergic P2X, P2y
cells
Brautigam et
al, 2006
HMGB1
RAGE
nonhistone
DNA-binding
protein
Kim et al, 2006
S100B
RAGE
calcium binding
protein
Bianchi et al,
2007
15d-PGJ2
PPAR
prostaglandin
Gurley et al
2007
Immunological Trigger
(LPS, gangliosides)
Neurotoxic Trigger
(Glutamate)
(Block and Hong, 2005)
ROS/RNS –
Reactive
Oxygen/Reactiv
e Nitrogen
Species
Abbreviation
Full name
Function/effect
IL-1β
Interleukin-1 beta
Proinflammatory
Il-6
Interleukin-6
Proinflammatory
TNF-α
Tumor necrosis factor alpha
Proinflammatory
NO
Nitric oxide
Neurotoxic
NOO-
peroxynitrite
Neurotoxic
O2-
superoxide
Neurotoxic
H2O2
Hydrogen peroxide
Neurotoxic
OH-
Hydroxyl radical
Neurotoxic
IL-10
Interleukin-10
Immunosuppressive
BDNF
Brain derived neurotrophic factor
Neurotrophic
NGF
Nerve growth factor
Neurotrophic
NT-3
Neurotrophin-3
Neurotrophic
(Block and Hong, 2005)
Neurotrophic factors: Also Ciliary Neurotrophic Factor (CNTF)
(Some evidence for) constitutive and (more evidence for) induced release (Heese et al, 1998, Nakajima et al,
2001)
Increase phagocytosis (Lee et al, 2009)
Ab-TLR4
TLR3
TLR4
Ab-TLR4
TLR2
Ab-TLR3
Ab-TLR2
PIC - Poly(inosinic acid):poly(cytidylic acid);
PAM - palmitoyl-3-cysteine-serine-lysine-4.
ALL FIGURES: Human microglia cell cultures
treated for 24 hours with ligands, supernatants
tested for cytokines with ELISA. n=3, +/-SEM.
Cell cultures pre-incubated 20 minutes with Ab before ligand treatment.
NOTE: TLR3 is predominantly intracellular in microglia (results not shown).
(Jack et al, 2005)
NO
Nitrite + Nitrate
Nitrate reductase
Nitrate
Nitrite +
Nitrite
1% sulphanilamide
0.1% N-(1-naphthyl) ethylenediamine-HCl
Read absorbance at 540, 570nm,
compare to standard curve of sodium
nitrite
Maximal stimulation by 2 agonists
Treatment with 2 ligands, each at 100% of
individual concentration needed to induce
maxium NO release. n=6, +/- STDEV
Sub-maximal stimulation
Treatment with 2 ligands, each at 10-30% of
individual concentration needed to induce maxium
NO release. n=4, +/- STDEV (n=3 for measurements
with HKAL)
Mice microglia cell cultures treated for 24 hours with indicated ligands.
Critique: nitrate not converted to nitrite. p values for supradditive compared
to 2 ligands not donel.
(Ebert et al, 2005)
Nitrite release
Neuron viability
Microglia/neuron
Microglia only
Neurons only
Rat microglia treated as indicated for 24 hours. B= Blue
G, a P2X7R antagonist. n=4 cultures +/- SD
Critique: LDH assay (rt) is not specific to neurons. Microglia
LDH subtracted from total, but does not allow for combined
effects.
Incubated as indicated for 72 hours. Rat microglia and
rat cortical neurons. OxATP (200μM) is a P2X
antagonist (not neurotoxic, pretreated for 60 minutes).
Lactate dehydrogenase (LDH) indicates cell viablity:
released only when membrane is disrupted. (n=3 exp, 3
cultures each, +/- SD)
(Skaper et al, 2006)
Time course of varying [LPS]-induced cytokine release
Viability of murine motor neurons
Left: BV-2 cells exposed to indicated LPS concentrations. Cytokines
quantified by ELISA. Above: NSC34 (murine motor neuron) cells
treated with LPS stimulated BV2 culture media. NSC34 cells
incubated for 36 hours with LPS-BV2 culture media. Viability
determined by MTS assay.
Critique: LPS-BVCM incubation time prior to supernatant collection
and application to neurons not furnished.
(Li et al, 2007)
P2Y / P2X
P2Y1
P2Y / P2X
P2Y1
P2
P2Y1
AGONISTS
ANTAGONISTS
P2X7
P2X1 / P2X3
Left and right: BV-2 microglia co-treated as shown for 20 hours. Nitrite production measured by Griess reaction (n= 3-5 +/SEM).
Critque: Details on quantities added to cell cultures, and determination of [LPS] not given. Nitrate not converted to nitrite in
Griess assay.
(Brautigam et al, 2005)
Cytokine expression
Motor neuron viability
NOTE: Rat microglia express CNTFRα (results not shown). Left: Microglia stimulated for 8 hours as indicated. Total
RNA was reverse transcribed and analyzed by rtPCR (n=3 +/-SEM). COX-2 protein levels showed similar response to
IL-6 and CNTF (data not shown). Right: Microglia treated with MN1a (medium), IL-6 or CNTF for 6 hours. Cells were
rinsed and incubated 2 days with MN1a. Culture media was then incubated with motor neurons for 2 days. Motor
neurons quantified with Ab against choline acetyltransferase (ChAT) (n=3 +/-SEM).
Critique: details on ChAT Ab lableled neuron counting not published
(Krady et al, 2008)
Left: Rat neutrophils and rat primary microglia added simultaneously to rat
organotypic hippocampal slice cultures (OHC) after oxygen– glucose
deprivation (OGD) (n=9, +/-SEM). RAW 264.7 = mourse macrophage line.
Cell death measured by propidium iodide (PI) incorporation into damaged
cells (red fluorescent signal).
Critque: PI not specific for neurons
(Neumann et al, 2008)
Left: primary rat microglia were incubated for 6 hours with LPS as indicated.
Neurotrophin secretion measured by Western blot. n=3, representative blot
shown. Above: NO and TNFα are secreted similarily to BDNF. Above: BDNF and
TNF-α measured by Western blot. NO measured by exclusive NO analyzer.
Values normalized to 6 hours. n=3 +/- SD Also found that BDNF and NGF are
secreted constitutively (not shown). BDNF secretion linked to PKC pathway.
Critique: graph of actual values in table 1 may have provided more information
(relative amounts of release).
(Nakajimai et al, 2001)
Authors
Finding used
Support
critique
Jack et al, 2005
Activation of TLR 2, 3 & 4 induces proinflammatory cytokine response in microglia
Partial
support (1)
none
Ebert et al, 2005
Submaximal stimulation of 2 TLRs results in
additive or supraadditive NO release
Partial
support (1)
Nitrate not converted to
nitrite. p value for
supradditive
Skaper et al, 2006
Activation of P2X7 receptor in rat microglia
results in neurodegenerative effects
Does not
support
(1)support(2)
LDH not specific to
neurons.
Li et al, 2007
Microglia secreted factors may be
neurotrophic or neurodegenerative
depending on degree of LPS stimulation
Support (1,2)
Incubation time of LPSBVCM not published
Brautigam et al,
2005
P2Y and P2X (including P2X7) receptors
reduce pro-inflammatory BV-2 microglia
response
Paritial
support (1)
Method details mssing.
Nitrate not converted to
nitrite.
Krady et al, 2008
Ciliary neurotrophic factor (CNTF) has antiinflammatory and neuroprotective effects
which are mediated by rat microglia
Support (1,2)
ChAT Ab labeled neurons
counting details not
published
Neumann et al,
2008
Rat Primary Microglia Have Neuroprotective
Effects Against Neutrophils
Support (2)
PI assay is not specific for
neurons – relies on prior
results w/o PMNs
Nakajimai et al,
2001
LPS induces release of neurotrophins in
primary rat microglia
Does not
support (1)
Present table as figure
Do microglia have a phenotypically distinct neuroprotective
activation state, as measured by cell surface receptor expression?
1) Differentially
activate microglia
Control
No activation
2) Check for
presence of cell
surface receptors
(flow cytometry)
Known activation markers: CD11b (const.), CD45, MHC1, MHC2, B71, B7-2, CD40, ICAM1, Fcγ (Olsen & Miller 2004, Kim and De Vellis, 2005)
3) Measure
secreted factors
TNF-α, IL-1β, IL-6, IL-10, BDNF, NO
(Western blot, ELISA, rtPCR, Griess Assay)
4) Incubate
neurons w/ culture
media
5) Measure neuron
survival
Neurodegenerative
High [LPS]
Neuroprotective
CNTF &/or low [LPS]
Receptors linked to anti-inflammatory phagocytosis: TREM-2, PS-R
(Neumann et al, 2008-2)
36 – 48 hours
(Li et al, 2007, Krady et al 2008)
MTS assay, PI incorporation
(Li et al, 2007, Neuman et al, 2008)