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

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

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

• 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?

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

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

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

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

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

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

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

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

?