1

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

2

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

3

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

4

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

5

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

6

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

8

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

9

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