1. Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia Berrih-Aknin
Muscle pathophysiology: Anti-AChR antibodies increase IL-6 production by myoblasts and myotubes in vitro
Sylvain Bougoin, Nicole Kerlero de Rosbo, Sonia Berrih-Aknin
UMR CNRS-UPS 8162, Centre chirurgical Marie Lannelongue, Le Plessis Robinson, France
International Conference on Myasthenia Gravis, December 1-2, 2009

2. Understanding the consequences of the autoimmune attack on the muscle function
Anti-AChR antibodies
MIR : Main Immunogenic Region
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

3. Transcriptome analysis of rat and human muscle
Rat / EAMG Rat
(Feferman, Fuchs, Souroujon)
Human
Control / MG
List of dysregulated genes
List of dysregulated genes
Muscle structural genes regulated
Genes coding for heat-shock proteins
Signaling pathways
Ex: IL-6
Abs effect on IL-6 production?

4. Why IL-6? 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)

5. 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
Human myogenic
cell line (LHCN)
(V. Mouly )
Myoblasts
Myotubes

6. Effects of patient sera on IL-6 production
Kinetics of IL-6 production
IL-6 production at 48 hours
p<0.01
p<0.05
Patient sera induce the production of IL-6 by myoblasts
Is the serum effect due to anti-AChR antibodies ?

7. Effects of monoclonal anti-AChR antibodies on IL-6 production
MAbs: monoclonal antibodies (Rat)
Kinetics of IL-6 production
IL-6 production at 48 hours
p<0.001
p<0.01
Anti-AChR antibody 198 induces the production of IL-6 by myoblasts
Is the IL-6 production due to AChR internalization?

8. Ab 198 induces both a strong internalization and IL-6 production
Effects of Mab anti-AChR on human myotubes
AChR expression at 12 hours
Kinetics of IL-6 production
p<0.01
Ab 198 induces both a strong internalization and IL-6 production

9. Effect of MG sera on surface AChR levels
AChR expression
Kinetics of IL-6 production
Poea S. et al, 2000
SNMG
SPMG
CONTROLS
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?

10. Work in progress: Signaling Pathways NF-κB… Ca2+ Release
10µM
Ca2+ Release
Signaling Pathways
NF-κB…
2µM
5µM
Il-6 production

11. IL-6 production A new action mechanism of anti-AChR on muscular cells
Conclusion:
Sera (Control, SPMG, SNMG)
Mabs (198, 155) (S. Tzartos)
Myotubes
Myoblasts
IL-6 production
A new action mechanism of anti-AChR on muscular cells

13. Future work
MG: Regenerative capacity of satellite cells
Ca2+ Release ? ?
Satellites cells proliferation
Il-6 production
Cyclin D1
JAK2/STAT3
Mc Kay et al. 2009 ?
Local immune role