wallerian degeneration the innate immune response of the pns...
TRANSCRIPT
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Wallerian degenerationthe innate immune response of the
PNS to traumatic injury
Macrophage and Schwann cell activation&&
the cytokine network of Wallerian degeneration
Shlomo Rotshenker
Dept. of Medical Neurobiology
Hebrew University Faculty of Medicine
PNS is regenerating - CNS is not
CNSnon‐
iaxotomy
PNSregeneratingPNS
regeneratingCNS
y
axotomy
CNS neurons regenerate axons throughWallerian degenerated PNS but not CNS tissue
David and Aguayo, 1981from Neuron to Brain, Nicholls et al., Sinauer 2001
PNS: (A) intact and (B) Wallerian degeneration
myelinatingSchwann
basal laminaA fibroblast
AXOTOMY
Bactivated macrophage & Schwann
PNS ‐WD
injury site
Wallerian degenerationAugustus Waller (1850)
Waller, Phil. Transact. Royal. Soc. London 1850
Intact PNS: myelinated axon
Schwann
axon
myelin
Reichert, Saada & Rotshenker, J. Neurosci. 1994
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Normal PNS Wallerian degeneration
50
60
70
80
90
100
110
onte
nt
Po MBP
macrophage
0
10
20
30
40
50
0 5 10 15 20 25 30
Days
% c
o
Schwann cells
Reichert, Saada & Rotshenker, J. Neurosci. 1994 Be’eri, Reichert, Saada & Rotshenker, Eur. J. Neurosci. 1998
Normal Wallerian degeneration
• Axons degenerate
• Schwann cells reject their myelin
• Schwann cells & fibroblasts proliferate
• Macrophages are recruited from circulation
• Macrophages & Schwann cells are activated to clear and degrade degenerated‐myelin
Galectin-3/MAC-2 marks macrophage and Schwann cell activation in normal Wallerian degeneration
70
80
90
100
110
t
Galectin-3/MAC-2
Schwann cells
0
10
20
30
40
50
60
0 5 10 15 20 25 30
Days
% c
onte
n
macrophages
Be’eri, Reichert, Saada & Rotshenker, Eur. J. Neurosci. 1998Reichert, Saada & Rotshenker, J. Neurosci. 1994
Schwann cell & macrophage activationmyelin phagocytosis & degradation
70
80
90
100
110
t
Galectin-3/MAC-2
70
80
90
100
110
t
Po MBP
0
10
20
30
40
50
60
0 5 10 15 20 25 30
Days
% c
onte
n
0
10
20
30
40
50
60
0 5 10 15 20 25 30
Days
% c
onte
nt
Be’eri, Reichert, Saada & Rotshenker, Eur. J. Neurosci. 1998
Schwann cell & macrophage activationmyelin phagocytosis & degradation
are orchestrated in time
70
80
90
100
110 Po
MBP
Galectin-3/MAC-2
0
10
20
30
40
50
60
70
0 5 10 15 20 25 30
Days
% c
onte
nt
Be’eri, Reichert, Saada & Rotshenker, Eur. J. Neurosci. 1998
slow Wallerian degeneration (slow-WD) in mutant Wlds (Ola) mice
myelinatingSchwann
basal laminaA fibroblast
B
proximaldomain
distal domain
slow ‐WD
injury site
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slow Wallerian degeneration in Wlds mice
slow Wallerian degeneration domain
injury domain
Reichert, Saada & Rotshenker, J. Neurosci. 1994
in-vitro degeneration in Wlds mice
in‐vivoslow Wallerian degeneration
in‐vitro degeneration
in‐vivo injury domain
Reichert, Saada & Rotshenker, J. Neurosci. 1994
PNS: (A) normal-WD and (B) slow-WD
AXOTOMY
Aactivated macrophage & Schwann
B
proximaldomain
distal domain
injury site
injury site
Events associated with Wallerian degeneration
• Normal regeneration • Delayed regeneration
normal ‐WD slow ‐WD
• NGF is upregulated
• Neuropathic pain –rapid development
• NGF is not upregulated
• Neuropathic pain –delayed development
Is there a single mechanism that underlies normal- and slow-Wallerian degeneration
and the events associated with them ?
Wallerian degeneration is the innate immune response of the PNS to traumatic axonal injury
• Normal‐WD results from and is a manifestation of a normal innate immune response
• slow‐WD results from and is a manifestation of a deficient innate immune response
Cytokines are the mediators of inflammation
Inflammatory – initiation and progression• TNFα – tumor necrosis factor α
• IL‐1α – interleukin‐1α
• IL 1β interleukin 1β• IL‐1β – interleukin‐1β
• IL‐6 – interleukin 6
• GM‐CSF – granulocyte macrophage colony stimulating factor
Anti‐inflammatory – down regulation• IL‐10 – interleukin‐10
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Cytokines in Wallerian degeneration
1. Mediators of inflammation
2. Activate Schwann cells and macrophages to clear degenerated‐myelin
3. Regulate NGF production
4. Nerve growth factors
5. Regulate neuropathic pain
Cytokine Network of Wallerian degeneration
AXOTOMY
TNFα
IL‐1αIL‐6 GM‐CSF
IL‐10
IL‐1
TNF IL 1 IL 1IL‐6
TNF IL‐1 IL‐1
TNF IL‐1 IL‐1
IL‐6GM‐CSF
IL 6
IL‐10
Studying cytokine production
Immune and non‐immune cells synthesize and secrete cytokines that bind and activate cognate receptors on immune cells
t i ti t ki RNA i• transcription – cytokine mRNA expression
• translation – cytokine protein in cells
• secretion – cytokine protein in extracellular space
• which cell type
TNF, IL-1 & IL-1 mRNA in normal Wallerian degeneration
Shamash, Reichert & Rotshenker, J. Neurosci. 2002
++
++
-
+
IL-1, IL-1 & TNF proteins in normal Wallerian degeneration
+++
+++
+++
Shamash, Reichert & Rotshenker, J. Neurosci. 2002
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TNF IL-1 & IL-1 protein in nerve derived non-neuronal cells
S-100 Gal-3 CR3 F4/80
++--Schwann
TNF IL‐1 IL‐1
-+++
----
macrophage
fibroblast
Shamash, Reichert & Rotshenker, J. Neurosci. 2002
IL-1, IL-1 & TNFmRNA in Schwann cells & macrophages
Shamash, Reichert & Rotshenker, J. Neurosci. 2002
IL-1, IL-1 & TNF proteinin slow Wallerian degeneration
injurydomain
slow‐WDdomain
Shamash, Reichert & Rotshenker, J. Neurosci. 2002
IL-1, IL-1 & TNF mRNA in slow Wallerian degeneration
Shamash, Reichert & Rotshenker, J. Neurosci. 2002
IL-1, IL-1 & TNFin slow Wallerian degeneration
+ + +
+ + +injurydomain slow-WD
domain
Shamash, Reichert & Rotshenker, J. Neurosci. 2002
GM-CSF production innormal and slow Wallerian degeneration
2.5
3.0
3.5
4.0rapid-WD
slow-WD
pg/m
l/5h
N D1 D5
0.3
0.4
exp
res
sio
n)
0.0
0.5
1.0
1.5
2.0
0 5 10 15
Days
GM
-CS
F p
0.0
0.1
0.2
Mac Fib Sch
Act
ivity
(G
ale
ctin
-3 e
Saada, Reichert & Rotshenker, J. Cell Biology 1996Be’eri, Reichert, Saada & Rotshenker, Eur. J. Neurosci. 1998
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IL-6 production in normal and slow Wallerian degeneration
0.8
1.0
1.2
1.4
g/m
g/5
h
rapid-WD
slow-WD
N D1 D5
5
6
7
8
9
h/24
h0.0
0.2
0.4
0.6
0 5 10 15
IL-6
ng
Days
0
1
2
3
4
5
Mac Fib Sch
IL-6
ng/
dish
Reichert, Levitzky & Rotshenker, Eur. J. Neurosci. 1996
IL-10 production innormal and slow Wallerian degeneration
14
16
18
20rapid-WD
slow-WD
N D1 D5
800
1000
1200
s
0
2
4
6
8
10
12
0 5 10 15
Days
IL-1
0 p
g/m
l/5h
0
200
400
600
800
Mac Fib Sch
IL-1
0 p
g/1
05ce
lls
Be’eri, Reichert, Saada & Rotshenker, Eur. J. Neurosci. 1998
Cytokine Network of Wallerian degeneration
AXOTOMY
TNFα
IL‐1αIL‐6 GM‐CSF
IL‐10
IL‐1
TNF IL‐1 IL‐1
normal Wallerian degeneration
TNF IL‐1 IL‐1
IL‐6GM‐CSF
IL‐6
IL‐10
TNF IL‐1 IL‐1
slow Wallerian degeneration
TNF IL‐1 IL‐1
IL‐6GM‐CSF
IL‐6
IL‐10
Cytokine production innormal Wallerian degeneration
Rotshenker, J. Neuroinflammation. 2011
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Macrophage recruitmentin normal Wallerian degeneration
Gitik, Reichert & Rotshenker, unpublished
Cytokine production innormal Wallerian degeneration
Rotshenker, J. Neuroinflammation. 2011
M1-type and M2-type macrophages
Ly6C/Gr1+
TNFα & IL‐1ROS IL‐6phagocytosiskilling
Ly6C/Gr1‐
IL‐10IL‐6phagocytosiswound‐healingtissue remodeling
Benoit, Desnues & Mege, J. Immunol. 2008Auffray, Sieweke, & Geissmann, Ann. Rev. Immunol. 2009
Are macrophages friend or foe?
Anti‐Inflammatory cytokines– IL‐10
Phagocytosis
M1 type – Killer
Inflammatory cytokines– TNFα, IL‐1β
Neurotoxicity
M2 type – Wound healing
Phagocytosis – apoptotic cells
– pathogens
– neurotoxic products of neurodegeneration
– tissue debris
Neurotoxicity– ROS
– PGE
Phagocytosis
Cytokine network ofWallerian degeneration Events associated with Wallerian degeneration
• Normal regeneration • Delayed regeneration
normal ‐WD slow ‐WD
• NGF is upregulated
• Neuropathic pain –rapid development
• NGF is not upregulated
• Neuropathic pain –delayed development
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Normal regeneration
Normal removal of myelinmacrophage recruitment and activationSchwann cell activationmyelin phagocytosis
Neurotrophic factors NGF
Fixed attraction moleculesSchwann cells, basal lamina, collagen
Cytokines regulate myelin removal
• TNF, IL‐1 and IL‐1 function as chemoattractant to recruit macrophages
• TNF, IL‐1 and IL‐1 up‐regulate production of macrophage chemoattractants MCP‐1 and MP‐1macrophage chemoattractants MCP 1 and MP 1
• GM‐CSF activates macrophages and Schwann cells to clear degenerated‐myelin by up‐regulatingGalectin‐3/MAC‐2
• GM‐CSF up‐regulates CR3/MAC‐1
• cytokines up‐regulate myelin phagocytosis
TNF IL‐1, IL‐1
MCP‐1activation/phagocytosis
GM‐CSF
MCP‐1
IL‐6
cytokines
activation
phagocytosis
NGF
TNF IL‐1 IL‐1
NGF
NGF
survival and regeneration ofsensory neurons & sympathetic neurons
Cytokines regulate neuropathic pain
• TNF, IL‐1 and IL‐1 produce spontaneous firing in sensory neurons
TNF IL 1 d IL 1 l t NGF d ti i• TNF, IL‐1 and IL‐1 up‐regulate NGF production in fibroblasts and in vascular endothelial cells. In turn, NGF produces neuropathic pain by sensitizing sensory nerve endings/receptors
• IL‐6 deficient mice display reduced neuropathic pain
NGF
TNF IL‐1, IL‐1
NGF
NGF
neuropathic pain
sensitization
intact intact
spontaneous firing
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Wallerian degeneration is the innate immuneresponse of the PNS to traumatic axonal injury
• Normal myelin removal • Delayed myelin removal
normal ‐WD slow ‐WD
• Normal regeneration
• NGF is upregulated
• Neuropathic pain –normal development
• delayed regeneration
• NGF is not upregulated
• Neuropathic pain –delayed development
TNF IL‐1 IL‐1
Normal Wallerian degeneration
TNF IL‐1 IL‐1
IL‐6GM‐CSF
IL‐6
IL‐10