Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in...
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Transcript of Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in...
![Page 1: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/1.jpg)
Muscle pathophysiology:Anti-AChR antibodies increase IL-6 production
by myoblasts and myotubes in vitro
Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia Berrih-AkninUMR CNRS-UPS 8162, Centre chirurgical Marie Lannelongue, Le Plessis Robinson, France
International Conference on Myasthenia Gravis, December 1-2, 2009
![Page 2: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/2.jpg)
MIR : Main Immunogenic Region
Anti-AChR antibodies
Understanding the consequences of the autoimmune attack on the muscle function
Accelerated degradation of AChR
(Drachman et al. 1977)
Complement mediated damage
(Engel et al. 1976)
Blocking antibodies (Howard et al. 1987)
MG Muscle
(Adapted from Drachman et al, 1994)
Control Muscle
![Page 3: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/3.jpg)
Transcriptome analysis of rat and human muscle
Transcriptome
Abs effect on IL-6 production?
Muscle structural genes regulated
Genes coding for heat-shock proteins
Signaling pathwaysEx: IL-6
Rat / EAMG Rat(Feferman, Fuchs, Souroujon)
HumanControl / MG
List of dysregulated genes List of dysregulated genes
![Page 4: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/4.jpg)
• Important role in the immune system: Differentiation of the B cells (Klashman DJ. et al, 1991)
Differentiation, proliferation and survival of lymphocytes (Smol'nikova V.V. et al, 2000)
• High IL-6 gene expression and production by cultured human thymic epithelial cells from patients with myasthenia gravis (Cohen-Kaminsky S. et al, 1993)
• IL-6 has a role in muscular contraction and is a regulator of Skeletal Muscle Hypertrophy (McKay R. et al, 2009; Serrano L. et al, 2008)
• No MG induction in IL-6-/- mice (Deng C. et al, 2002)
Why IL-6?
![Page 5: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/5.jpg)
Human myogenic cell line (LHCN)
(V. Mouly )Myoblasts Myotubes
Methods
- Internalisation (Alpha-Bgt binding)- IL-6 production (ELISA assays)
- Sera (Control, SPMG, SNMG)- Mabs (198, 155) (S. Tzartos)
Mab 198
Mab 155
Extracellular
Intracellular
![Page 6: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/6.jpg)
0 20 40 600
20
40
60
80
SPMG (n=8)Control (n=8)
SNMG (n=6)
Time (hours)
pg
/ml
Effects of patient sera on IL-6 production
Control SPMG SNMG0
50
100
150
pg
/ml
Patient sera induce the production of IL-6 by myoblasts
Is the serum effect due to anti-AChR antibodies ?
Kinetics of IL-6 production IL-6 production at 48 hours
p<0.01
p<0.05
![Page 7: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/7.jpg)
Effects of monoclonal anti-AChR antibodies on IL-6 production
MAbs: monoclonal antibodies (Rat)
IgG2a 198 IgG1 1550
20
40
60
80
100198IgG2a155IgG1
IL-6
pg
/ml
Anti-AChR antibody 198 induces the production of IL-6 by myoblasts
IL-6 production at 48 hours
0 20 40 600
20
40
60
80
100198 IgG2a155IgG1
Time (hours)
IL-6
pg
/ml
Is the IL-6 production due to AChR internalization?
Kinetics of IL-6 production
p<0.001 p<0.01
![Page 8: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/8.jpg)
155 19820
40
60
80
100
AC
hR a
lpha
-Bgt
bin
ding
site
(%
)
0 20 40 600
20
40
60
80
198IgG2a155IgG1
Time (H)p
g/m
l
AChR expression at 12 hours
Effects of Mab anti-AChR on human myotubes
Ab 198 induces both a strong internalization and IL-6 production
Kinetics of IL-6 production
p<0.01
![Page 9: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/9.jpg)
Effect of MG sera on surface AChR levels
0 20 40 600
50
100
150
200ControlSPMGSNMG
Time (hours)
pg
/ml
Surprisingly, sera from patients with anti-AChR antibodies do not induce significant production of IL-6, while sera with undetectable antibodies do
Production of IL-6 and internalization appear to be independent events
Mechanisms involved?
Kinetics of IL-6 productionAChR expression
Poea S. et al, 2000
SNMG SPMGCONTROLS
![Page 10: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/10.jpg)
Work in progress:
Ca2+ ReleaseSignaling Pathways
NF-κB…
Il-6 production
Ab-198
IgG2a
PDTC
0
50
100
150
200
250
300
350
% IL
-6 p
rod
uct
ion
10µM
Ab-198
IgG2a
Mib
efra
dil
Nifédin
pin
50
100
150
200
250
300
350
% IL
-6 p
rod
uct
ion
2µM 5µM
![Page 11: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/11.jpg)
Conclusion:
Myoblasts Myotubes
IL-6 production
- Sera (Control, SPMG, SNMG)- Mabs (198, 155) (S. Tzartos)
A new action mechanism of anti-AChR on muscular cells
![Page 12: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/12.jpg)
![Page 13: Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia.](https://reader035.fdocuments.in/reader035/viewer/2022062515/56649f575503460f94c7c320/html5/thumbnails/13.jpg)
Future work
Il-6 production
?
Satellites cells proliferation
Cyclin D1JAK2/STAT3Mc Kay et al. 2009
MG: Regenerative capacity of satellite
cells
Ca2+ Release?
Local immune role
?