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The Sympathetic Nervous System (SNS)
A not so “sympathetic”
regulator of immune
function in autoimmune
disease:RA as an example
Kent State University‡
Loma Linda University¤
Dianne Lorton ‡
Denise Bellinger ¤
3rd International Conference on Clin,
& Cell. Immunol, Sept 23- Oct. 1, 2014
Blood Borne
SignalsSympathetic
Nervous
Neural-Immune Cross-Talk
SignalsTNF, IL-1, IL-6
Bone
Marrow
Thymus
Spleen
BloodGALT
Lymph
Node
Lymphocytes
Macrophages
Dendritic cells
Others
Pathogens DiseaseX
Nervous
System
� Autoimmune Disease
� Chronic inflammatory response
� Production of autoantibodies
� Loss of Tolerance: Imbalance
between autoreactive effector T cells
Rheumatoid Arthritis
(CD4+ Th1 & Th 17) and T reg cells
� Th cell balance regulated by the SNS
� SNS activity is chronically elevated in RA patients
� How this impacts Th cell balance is not known
T ββββ2222
CD4+ ββββ2 (200-750 sites/cell)
CD4+ Th1 clones ββββ2 (250 sites/cell)
SNS Regulates Th Cell Differentiation via β2-AR
Activation of cAMP-PKA Pathway
.L
ACGs
nerve
terminal
B
APCs
αααα, αααα1111, α, α, α, α2222, β, β, β, β, β, β, β, β2222
CD4+ Th1 clones ββββ2 (250 sites/cell)
CD4+ Th2 clones (no detectable ββββ2)
CD4+ Treg cell 1?
CD4+ Th17 cells?
ACGs3
β2-AR
G –protein
Adenylate cyclase
Lipid Raft
Gs
ATP cAMP5’ AMP
PKA
AC
1Guereschi et al. Eur J Immunol. 2013 Apr;43(4):1001-12.
β2-AR Shifts Th0 cell →Th2 Differentiation
TNF-α, IL-12
IFN-γ, IL-2
Th0-CellTh1
Th1-Cell
Th2-Cell
APCMicrobial
Viral
β2-ARαααα-AR
Tregs?
Th17?
IL-10 IL-4
Th2-Cell
Th2Th0-Cell
Th2-Cell
APCParasite
Allergen
Guereschi et al., 2013
Hypothesis: Reduce disease severity is due in
part to a β2-AR driven shift in Th1 vs Th2 cell
balance.
Disease Onset
Ag Processing/
Clearance
Effector
Phase
Challenge
Joint
InflammationClearance Phase
increasing severity
ββ22--AR agonistAR agonistss
Inflammation
(AA: Lorton et al., 1998; 2004)
(CIA: Malfait et al., 1999; Härle et al., 2005) AA, Terbutaline D12-28
Day 0Terbutaline (β -AR agonist;
Day 12-28
Experimental Design
Adjuvant-Induced
Arthritis (AA)
CFA (0.3 mg M. butyricum
in 100 µµµµl MO)
Terbutaline (β2-AR agonist;
1.5 mg/ml/day i.p.)
Saline Vehicle
Outcome AssessmentsTh1/2 cell cytokines (ELISAs)
Foot Pad Swelling (data not shown)
X-ray analysis (data not shown)
Day 28
Draining Lymph
Nodes (DLN)
Spleen
PBMCs
100
200
300
400
500
IFN
- γγ γγ(p
g/m
l)Spleen: Failure of a β2-AR agonist to shift
from a Th1 to Th2 cytokine profile
50
100
150
200
250
300
TN
F-α
(pg
/ml)
A. B.
No Change in IL-4; (40-80 pg/ml)
0
VEH TERB
0
10
20
30
40
50
VEH TERB
IL-2
(p
g/m
l)
0
200
400
600
800
1000
VEH TERB
IL-1
0 (
pg
/ml)
*
0
VEH TERB
Anova with Bonferoni post-hoc test, N = 8
C. D.
75
150
225
300
375
450
IFN
- γγ γγ(p
g/m
l)
*
0
50
100
150
200
250
TN
F-α
(pg
/ml)
DLN: β2-AR agonist promotes a Th1
cytokine profileA. B.
0
VEH TERB
No Change in IL-4 (40 -80 pg/ml)
0
VEH TERB
0
10
20
30
40
50
60
70
VEH TERB
IL-2
(p
g/m
l)
T
0
50
100
150
200
250
VEH TERB
IL-1
0 (
pg
/ml)
C. D.
Anova with Bonferoni post-hoc test, N = 8; *P<0.05
0
20
40
60
80
100
120
140
VEH TERB
TN
F-α
(pg
/ml)
0
100
200
300
400
500
600
VEH TERB
IFN
- γγ γγ(p
g/m
l)PBMC: Failure of a β2-AR agonist to shift
Th1 cytokine profilesA. B.
VEH TERB
0
20
40
60
80
100
120
140
VEH TERB
IL-2
(p
g/m
l)
0
10
20
30
40
50
VEH TERB
IL-1
0 (
pg
/ml)
VEH TERB
Anova with Bonferoni post-hoc test, N = 8; *P<0.05No Change in IL-4 (40 -80 pg/ml)Wahle et al., 2006. Failure of catecholamines to shift T-cell cytokine responses toward
a Th2 profile in patients with rheumatoid arthritis. Arthritis Res. Ther., 8(5):R138.
C. D.
Conclusions� Different responses in each tissue examined: animal
models critical for understanding RA
� Stimulating β2-ARs after disease onset fails to
inhibit Th1 cell driving cytokines
� Spleen: β2-AR agonists produced no change IFN-γ, IL-2,
α� Spleen: β2-AR agonists produced no change IFN-γ, IL-2,
IL-4 or TNF-α, and increased IL-10 (source ?)
� DLN stimulating β2-ARs promotes IFN-γ & IL-2, no
change in IL-4, IL-10, TNF-α
� β2-AR stimulation under normal circumstances
inhibits IFN-γ and IL-2 production via cAMP-PKA
� These findings indicate abnormal β2-AR functions
Conclusions
� In spleen cells, the inability of terbutaline to reduce
IFN-γ and IL-2 could be easily explained by the
well-known down-regulation and desensitization of
β2-AR with repeated stimulation.
� Subsequent, cAMP assays and receptor binding � Subsequent, cAMP assays and receptor binding
experiments, confirmed this hypothesis (Lorton et
al., Clin Dev Immunol., 2013)
� However, the terbutaline-induced increase in IFN-γand IL-2 were intriguing. � not explained by
canonical signaling of β2-AR
Does Altered β2-AR Coupling to Second Messengers
Occur in DLNs in AA: cAMP-PKA to ERK1/2?
β-Arrestin-dependent, G Protein-
independent ERK1/2 Activation by the β2
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 2, pp. 1261–1273, January 13, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Adrenergic Receptor*
Received for publication, June 16, 2005, and in revised form, November 2, 2005 Published,
JBC Papers in Press, November 9, 2005, DOI 10.1074/jbc.M506576200
Sudha K. Shenoy‡1,Matthew T. Drake‡2, Christopher D. Nelson‡, Daniel A. Houtz‡,
Kunhong Xiao‡, Srinivasan Madabushi§, Eric Reiter‡¶, Richard T. Premont‡, Olivier
Lichtarge§, and Robert J. Lefkowitz‡3
GRK5/6
P3
ACGs3P 3
PKA 6b
5
Altered Receptor Signaling in the DLN?
.L
nerve terminal
x
.L
ACGs3
ATP cAMP
L
nerve terminal
.
Gene Regulation
ERK1/2
MAPK
PP
ββββ-Arrestin
ATP cAMP
NoncanonicalCanonical
Gene Regulation
PKA
Transcription Factors Transcription Factors
CFA (0.3 mg M. butyricum
in 100 µµµµl MO)Mineral Oil (MO)
M. Butyricium (in saline;
SMB)
Saline
Day 1
Terbutaline (β -AR agonist;
Day 12-28
Hypothesis: Terbutaline induces a shift in β2-ARs
signaling from cAMP-PKA to ERK 1/2 in the DLN
Adjuvant-Induced
Arthritis (AA)
Saline Terbutaline (β2-AR agonist;
1.5 mg/ml/day i.p.)
Saline Vehicle
Outcome AssessmentsDLN: ββββ2-AR Western Blots
(antibodies to detect β2-ARs,
and β2-ARs phosphorylated by
PKA and or GRK)
Day 21 or 28
Draining Lymph
Nodes (DLN)
Unchanged DLN β2-AR Density Late Disease
0.4
0.6
0.8
1.0
No
rma
lize
d β
2-A
RT
(O.D
.)
0.4
0.6
0.8
1.0
No
rma
lize
d β
2-A
R
(OD
)
***
***A. D21 B. D28
5Cββββ2-ARDay 28
Saline MO SMB CFA
0.0
0.2
0.4
Saline MO SMB CFA
No
rma
lize
d
(O.D
.)
0.0
0.2
0.4
Saline MO SMB CFA
No
rma
lize
d
(OD
)
P < 0.05; P < 0.01, P<0.001ANOVA; Bonferoni Post-Hoc Test * ***N=4; **
Altered Receptor Signaling in the DLN?
PKA
P3
.L
ACGs3
nerve terminal
xDesensitization
GRK2
P
Conclusions: These findings along with increased IFN-γγγγ indicate
that β2-ARs in DLN are NOT down-regulated or desensitized.
P
Recycled to
membrane Degraded
or
P
ββββ-Arrestin
L
0.0
0.4
0.8
1.2
1.6
2.0
2.4
Saline MO SMB CFA
β2-A
RP
KA:β
2-A
RT
Ra
tio
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Saline MO SMB CFA
β2-A
RP
KA:β
2-A
RT
Ra
tio
***** **
**
β2-AR phosphorylated by PKA and GRK in DLN
A. D21 B. D28
P < 0.05; P < 0.01, P<0.001ANOVA; Bonferoni Post-Hoc Test * ***N=4; **
Saline MO SMB CFA Saline MO SMB CFA
0.0
0.3
0.6
0.9
1.2
1.5
Saline MO SMB CFA
β2-A
RG
RK
:β2-A
RT
Ra
tio
0.0
0.2
0.4
0.6
0.8
Saline MO SMB CFA
β2-A
RG
RK
:β2-A
RT
Ra
tio
***** **
*
*
*****C. D21 D. D28
GRK5/6
P3
ACGs3P 3
PKA 6b
5
Altered Receptor Signaling in the DLN?
L
nerve terminal
xPKA
P3
L
ACGs3
nerve terminal
or
xDesensitization
GRK2
P
Gene Regulation
ERK1/2
MAPK
PP
ββββ-Arrestin
Transcription Factors
P
Recycled to
membrane Degraded
or
P
ββββ-Arrestin
L
Conclusions: These findings coupled with increased IFN-γγγγ,
provide support β2-AR signaling via ERK1/2.
Summary
� Findings support a shift in β2-AR receptor
signaling from cAMP-PKA to ERK1/2 in
DLN
• β2-AR agonist elevated IFN-γ and IL-2• β2-AR agonist elevated IFN-γ and IL-2
• No change in β2-AR density,
• Receptor phosphorylation by PKA increased
PKA (day 21) and GRK phosphorylation (day 21
and 28)
Future Studies
• Are GRK5/6 and ERK 1/2 elevated in DLN cells?
• Can production of IFN-γ be blocked by inhibitors of
ERK1/2 pathway?
• Why the different profiles in the spleen and DLN?• Why the different profiles in the spleen and DLN?
– Inflammatory cytokine levels
– CFA distribution/concentration
• Does the SNS regulate balance between Th17
and Treg cells?
Acknowledgements
Cheri Lubahn, Ph.D.
Jill Schaller Tracy Osredkar
Kent State University
Loma Linda UniversitySchool of Medicine
Denise Bellinger, Ph.D.
Christine Molinaro
Funded by: NIMH, NIAMS, Arizona Disease CRC, Sun Health Research Institute &Sun City West Community Center Fund, LLU Anatomy and Pathology Dept.
SNS Function:
Respond to stress
&
maintain normal
body functions
(homeostasis)
The SNS
integrates the
functions of many
systems required
to mount an
immune response
Bone
T ββββ2222CD4+ ββββ2 (200-750 sites/cell)
CD8+ ββββ2 (500-2500 sites/cell)
CD4+ Th1 clones ββββ (250 sites/cell)
SNS Inhibition of Th1 Cytokines (IFN-γ and IL-2)
to Push Th2 Cell Differentiation Occurs via β2-AR
of cAMP-PKA Pathway
.L
ACGs
nerve
terminal
B
Monocyte/
macrophage
αααα, αααα1111, α, α, α, α2222, β, β, β, β, β, β, β, β2222αααα, αααα1111, α, α, α, α2222, β, β, β, β, β, β, β, β2222
CD4+ Th1 clones ββββ2 (250 sites/cell)
CD4+ Th2 clones (no detectable ββββ2)
CD4+ Treg cell 1
CD4+ Th17 cells?
ACGs3
β2-AR
G –protein
Adenylate cyclase
Lipid Raft
Gs
ATP cAMP5’ AMP
PKA
AC
1Guereschi et al. Eur J Immunol. 2013 Apr;43(4):1001-12.
The SNS
integrates the
functions of many
systems required
to mount an
immune response
Function:
Bone
Function:
Respond to stress
&
maintain normal
body functions
(homeostasis)
(allostasis- a new
“adaptive”
normal)
Reciprocal Immune System to
SNS Communication in
RA
n
Circulating
TNF-αααα. IL-1,
IL-6 RVLM
Hypothalamus
Cerebral Cortex
(Insula, limbic)
Stress
� Mechanism for
emotional distress to
impact health & disease n
Spleen
Pre-ganglionic
nerves
Joints
Lymph nodesimpact health & disease
� ~ 80% of patients
associate disease onset
with a severe emotional
life stressor (Trigger?)
� Stroke Victims: no RA in
paralyzed limbs (↓↓↓↓ vs ↑↑↑↑SNS nerve activity)
SNS
Saline Rx
0
500
1000
1500
2000
2500
3000
3500
4000
4500 4497Bmax =
Kd = 12.08
CP
M
0 1000 2000 3000 4000 50000
100
200
300
400Scatchard
Bound
Bo
un
d/F
ree
SMB Rx
0
1000
2000 Bmax =Kd =
2023
8.216
cp
m
0 500 1000 1500 2000 25000
50
100
150
200
250
Scatchard
Bound
Bo
un
d/F
ree
Splenocyte β2-AR Receptor Binding in Arthritic
Rats: Saturation Curves
0 25 50 75 100 1250
[I125
]CYP (pM)
0 25 50 75 100 1250
[I125
]CYP (pM)
mineral oil Rx
0 25 50 75 100 1250
1000
2000
3000
4000
5000 5405Bmax =Kd = 25.82
[I125
]CYP (pM)
cp
m
0 1000 2000 3000 4000 5000 60000
100
200
300
Scatchard
Bound (cpm)
Bo
un
d/F
ree
CFA Rx
0 25 50 75 100 1250
500
1000
1500
2000
2500
3000
3500Bmax =Kd =
382823.41
[I125] CYP (pM)
cp
m
0 1000 2000 3000 4000 50000
100
200
Scatchard
Bound (cpm)
Bo
un
d/F
ree
SNS-IS Cross-Talk Pathology in RA:
Reduced Spleen β2-AR Density Late Disease
0.6
1.0
No
rma
lize
d β
2-A
RT
(O.D
.)
0.4
0.6
0.8
1.0
No
rma
lize
d β
2-A
RT
(O.D
)
**
* *** * **A. D21 B. D28
Lorton et al., (2013) Clin Dev Immunol.;2013:764395P < 0.05; P < 0.01, P<0.001
ANOVA; Bonferoni Post-Hoc Test
* ***N=4; **
-0.2
0.2
Saline MO SMB CFA
No
rma
lize
d
0.0
0.2
Saline MO SMB CFA
No
rma
lize
d
(O.D
)
β2-AR Phosphorylation Patterns in the
Spleen
0.0
0.4
0.8
1.2
1.6
Saline MO SMB CFA
β2
-AR
PK
A:β
2-A
RT
Ra
tio
0.0
0.4
0.8
1.2
1.6
Saline MO SMB CFA
β2-A
RP
KA:β
2-A
RT
Ra
tio
**** * *** *
P < 0.05; P < 0.01, P<0.001
ANOVA; Bonferoni Post-Hoc Test
* ***N=4; **
Saline MO SMB CFA
0.0
0.2
0.4
0.6
0.8
1.0
Saline MO SMB CFA
β2-A
RG
RK
:β2-A
RT
Ra
tio
Saline MO SMB CFA
0.0
0.4
0.8
1.2
1.6
2.0
2.4
Saline MO SMB CFA
β2-A
RG
RK
:β2-A
RT
Ra
tio
****** **** *
Hypothesis: Chronic high SNS activity in RA induces
β2-AR down regulation and desensitization
aCFA/ICA (vehicle)
Autoantigen: HSP 65
Day 1a0.3 mg Mycobacterium butyricum
in 0.1 ml sterile mineral oil
Day 28
Day 1
cAMP assay
ββββ2-AR Receptor Binding Assays
ββββ2-AR Western Blots
using antibodies to detect
phosphorylated receptor
Day 21 or 28Harvest Spleen & DLN cells
AA
Day 28
Day 14
Day 28
Veh AA
Adjuvant-Induced Arthritis Rat Model
Day 1
Day 14
Day 1 Veh AA
Day 28
A B C
D
E
Day 28CFA (0.3 mg M. butyricum in
100 µµµµl MO)
0 3 7 12 21 28 Days
Autoreactive
T cells in
DLNs
Disease
InductionDisease
Onset
Autoreactive
T cells in
spleen
Chronic
Disease
Peak
Disease
F
SNS-IS Cross Talk in the Spleen?
ß2-AR & Signaling via the Canonical Pathway:
cAMP
.L
ACGs
nerve
terminal
P3
.L
ACGs
3
nerve terminal
xP
L
��� � ��
3
G –protein
Adenylate cyclase
Lipid Raft
Gs
ATP cAMP5’ AMP
PKAAC
Hypothesis: Chronic high SNS activity in RA induces β2-AR
down regulation and desensitization in splenocytes
PKA
P
P
Dephosphorylated
Recycled to membrane
Transported
to lysosome
Degraded
or
xDesensitization
GRK2
P
P
CFA (0.3 mg M. butyricum
in 100 µµµµl MO)Mineral Oil (MO)
M. Butyricium (in saline;
SMB)
Saline
Day 1
Terbutaline (β -AR agonist;
Day 12-28
Hypothesis: Chronic high SNS activity in RA induces
β2-AR down regulation and desensitization in the spleen
Saline Terbutaline (β2-AR agonist;
1.5 mg/ml/day i.p.)
Saline Vehicle
Outcome AssessmentsSpleen: cAMP assay (spleen)
ββββ2-AR Binding Assays
DLN: ββββ2-AR Western Blots
(antibodies to detect β2-ARs)
Day 21 or 28
Spleen
-6cell
s)
250
300
350
NoRx
Forskolin
Isoproteronol* * *
SNS-IS Cross-Talk Pathology in RA:
β2-AR Agonist Fails to Induce cAMP in SplenocytescA
MP
(fM
ol/
2 x
10
-
Non-AA AA
0
50
100
150
200
250 Isoproteronol
*
Anova with Bonferoni post-hoc test,
Day 28; N = 8; *P<0.05Lorton et al., (2013) Clin Dev Immunol.;2013:764395
SNS-IS Cross-Talk Pathology in RA:
Splenocyte β2-AR have Reduced Agonist
Affinity and Density
20
25
Kd
(I
CY
P (
pM
))
# # 1000
*
#
A. B.
Mineral Oil
Saline SMB CFA0
5
10
15
Kd
(I
CY
P (
pM
))
Saline Mineral Oil
SMB CFA
0
250
500
750
Bm
ax (
sit
es/c
ell)
*
P < 0.05; P < 0.01
ANOVA; Bonferoni Post-Hoc Test
* #N=6; Lorton et al., (2013) Clin Dev Immunol.;2013:764395
CFA (0.3 mg M. butyricum
in 100 µµµµl MO)Mineral Oil (MO)
M. Butyricium (in saline;
SMB)
Saline
Day 1
Terbutaline (b2-AR agonist;
Day 12-28
Hypothesis: Chronic high SNS activity in RA induces
β2-AR down regulation and desensitization in the spleen
Saline Terbutaline (b2-AR agonist;
1.5 mg/ml/day i.p.)
Saline Vehicle
Outcome AssessmentsSpleen: cAMP assay (spleen)
ββββ2-AR Binding Assays
DLN: ββββ2-AR Western Blots
(antibodies to detect β2-ARs)
Day 21 or 28
Draining Lymph
Nodes (DLN)Spleen
Altered Receptor Signaling in the DLN?
GRK5/6
P3
.L
ACGs3
nerve terminal
P 3
PKA6b
��� �
�� ��
PKA
P3
.L
ACGs3
nerve terminal
xDesensitization
↑↑↑↑↑↑↑↑↑↑↑↑ activity
��� �
�� ��
GRK2
P
Transcription Factors
Gene Regulation
ERK1/2
MAPK
5
PP
P
Dephosphorylated
Recycled to membrane Transported to lysosome
Degraded
or
Canonical
P
ββββ-Arrestin
L