relevance of autoantibodies in ra - euro diagnostica wj huizinga... · tom huizinga malmo 2012 ....
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Relevance of Autoantibodies in RA
Tom Huizinga
Malmo 2012
Quiz: How much of a patient IgG is
ACPA in the peripheral blood?
a) 0.005%
b) 0.05%
b) 0.5%
c) 5%
d) 50%
Amount of ACPA producing B cells
Average IgG Average ACPA Average ACPA ACPA of IgG IgG producing B cells produce ACPA
ng/ml U/ml ng/ml % 1 in x
PP2.60 773 0.155 3.714 0.48% 208
PP2.61 1227 0.393 9.438 0.77% 130
PP2.63 248 0.052 1.246 0.50% 199
PP2.66 454 0.074 1.782 0.39% 255
ACPA production by unstimulated PBMC’s
Assuming: ACPA producing B cells produce IgG
(ACPA) with the same rate as other B cells produce
IgG. PBMC/SFMC isolated with ficoll. Culture for +-2
weeks in IMDM 10% FCS
Quiz: How much of a patient IgG is
ACPA in the synovial fluid?
a) 0.005%
b) 0.05%
b) 0.5%
c) 5%
d) 50%
Amount of ACPA producing B cells
Average IgG Average ACPA Average ACPA ACPA of IgG IgG producing B cells produce
ACPA
ng/ml U/ml ng/ml % 1 in x
PP2.66
1253 14.7 353 28% 3.5 PP2.69
152 0.229 5.50 3.7% 28 PP2.72
2507 13 315 12 % 8
ACPA production by unstimulated SFMC’s
Outline:
- Antibodies to post translational modifications
- ACPA epitope recognition profile (Fine-specificity)
- Relevance of isotypes?
- Quantification per cell per time-unit
Outline:
- Introduction; ACPA; IgE
- Homo-citrulline vs citrulline; Can ACPA discriminate?
- Anti-Carbamylated Protein Antibodies (anti-CarP
antibodies)
- Anti-CarP-antibodies in health and disease
Autoantibodies and
immune complexes
Chronic Inflammation
and bone erosions
Arthritis
- Are specific for RA
- Are predictive for progression
towards RA
- Are directed against citrullinated
protein antigens that are also
expressed in the inflamed joint
- Have been shown to exacerbate
arthritis in mice
- Are predictive for a more severe
disease course
Anti-Citrullinated Protein Antibodies (ACPA)
ACPA positive
ACPA negative
Genetics
Environment
Progression
Remission
Severity
Differences in ACPA pos vs ACPA neg RA
ACPA+ RA ≠ ACPA- RA
ACPA positive
ACPA negative
Genetics
Environment
Progression
Remission
Severity
Differences in ACPA pos vs ACPA neg RA
ACPA+ RA ≠ ACPA- RA
Anti-MCV
Anti-CCP-2
Anti-CCP-3
Citr. Fibrinogen
Citr. Fibronectin
Citr. Vimentin
Citr. MBP
Citr. Enolase
Citr. Collagen
Et cetera
Anti-Citrullinated Protein Antibodies (ACPA)
recognize various citrullinated substrates
REACTIVITY TO CITR. MBP
Cit
r. M
BP
MB
P
Cit
r. M
BP
MB
P
Serum Eluate
REACTIVITY TO CITR. FIBRINOGEN
Cit
r. F
ibr
fib
r
Cit
r. F
ibr
fib
r
Serum Eluate
Anti-CCP-antibodies are ACPA and recognize
different citrullinated proteins
ACPA-positive serum
CCP column
Isolation of anti-Cit-fibrinogen
antibodies
Extensive cross-reactivity of ACPA towards
different citrullinated proteins
Ioan-Facsinay et al. Ann Rheum Dis 2011
Isolation of anti-Cit-MBP
antibodies
ACPA-fine-specificity
Vimentin
peptide B
Fibrinogen
peptide A
Enolase
peptide Fibrinogen
peptide B
Vimentin
peptide A
cVim1-16
cVim59-74
cFib α 36-52
cFib β 27-43
cEno5-20
cMBP
MCV
CCP3
53 subgroups
Willemze et al. Ann Rheum Dis 2011
ACPA-fine-specificity and „subgrouping“ of
patients (using 8 peptides)
Epitope recognition does not associate
with clinical characteristics
Willemze et al. Ann Rheum Dis 2011
cVim1-16
cFibα
cEno5-20
cVim59-74
cMBP
cFibβ
CCP3
MCV
RF
ANF
CCP2level=25 AU CCP2level >1000 AU
CCP2 level
Higher anti-CCP-2 levels associate with
enhanced epitope recognition
vimentin, enolase, fibrinogen,
collagen, fillagrin, EBNA-1, ...
no specific fine specificity that
associates with radiographic progression
or other clinical phenotypes Scherer et al, ARD 2011; Willemze et al, A&R 2011;
Willemze et al, ARD 2012
epitope spreading before disease onset,
then stable vd Woude et al, ARD 2010
RF
ANF
cVim1-16
cFibα
cEno5-20
cVim59-74
cMBP
cFibβ
CCP3
MCV
CCP2 level
Epitope recognition is correlated with
ACPA levels Willemze et al, ARD 2012
Biology of anti-citrulline immune responses;
fine-specificity
All isotypes are present; usage
broadens before disease onset Verpoort et al, A&R 2006; Ioan-Facsinay, A&R
2009; Van der Woude, ARD 2010
IgG1 99%
IgG2 82%
IgG3 60%
IgG4 98%
IgM 61%
IgA 62%
IgE 80%
Avidity of ACPA is low as compared
to anti-recall responses Suwannalai et al, ARD 2011; Suwannalai et al.,
A&R 2011
Biology of anti-citrulline immune responses;
isotype-usage and avidity
A broader isotype usage is associated with Radiographic
progression
* comparison ≤4 isotypes versus ≥5 isotypes: p<0.05
EAC-Netherlands EURIDISS-Norway
IgE is associated with immunity against
parasitic infections
The presence of IgE to allergens is classically
measured by a “Basophil Activation Test”
IgE Ag
IgE
20 min
37°C On ice Anti-CD63
CD63
FcεRI positive cell (mast cell/ basophil)
Allergen
IgE
Fc epsilon RI
Citr. Ag
IgE
Fc epsilon RI
Are FceRI-positive cells from ACPA-positive
patients activated by citrullinated antigens?
Anti-IgE Citr. Ag
IgE
20 min
37°C On ice Anti-CD63
CD63
Direct activation of IgE-positive basophils by
citrullinated Antigens?
Schuerwegh et al. PNAS 2010
0
25
50
75
100
aIgE PAD NC-FB C-FB-
ba
so
ph
il a
cti
va
tio
n%
of
ba
so
ph
ils
wit
h C
D6
3
up
reg
ula
tio
n
ACPA-positive RA
Direct activation of IgE-positive basophils by
citrullinated Antigens
Schuerwegh et al. PNAS 2010
ACPA-negative RA
0
25
50
75
100
- aIgE PAD NC-FB C-FB
ba
so
ph
il a
cti
va
tio
n
November 24, 2011 27
IL-8
IL-8
(n
g/m
L)
Ctr
Cit-Fib
Fib
1
2
3
4
25
15
aIgE
CD
20
3c-A
PC
CD63-PE
104
103
102
101
100
100 10
1 10
2 10
3 10
4
Ctr
CD
20
3c-A
PC
CD63-PE
104
103
102
101
100
100 10
1 10
2 10
3 10
4
CD
20
3c-A
PC
CD63-PE
104
103
102
101
100
100 10
1 10
2 10
3 10
4
104
103
102
101
100
100 10
1 10
2 10
3 10
4
aIgE Fib Cit-Fib
CD
20
3c-A
PC
CD63-PE
Degranulation
% C
D6
3+
ma
st ce
lls (
de
lta)
Ctr
aIgECit-Fib
Fib
10
20
30
40
50
*
*
Mast cells exposed to ACPA+ RA serum are
activated by Citrullinated proteins
Ctr aIgE Fib Citr. Fib
All patients
N=50
ACPA
positive
N=27
ACPA
negative
N=23
p-value
CD117 +
Mast Cells (MC) 27 (0-178) 33 (0-178) 11 (0-100) 0.003
degranulated MC
CD117+ CAE- 21 (0-173) 28 (0-173) 6 (0-67) 0.005
% degranulated MC 82 (0-100) 89 (0-100) 58 (0-100) 0.03
Increased number of degranulated mast cells
in synovial tissue of ACPA+ RA-patients
Schuerwegh et al. PNAS 2010
0
500
1000
1500
2000
2500
ACPA + RAACPA - RAOA
p< 0.0001
p= 0.008
D M
FL
Ig
E e
xp
ress
ion
Elevated IgE expression on ACPA+ Mast Cells
Schuerwegh et al. PNAS 2010
• ACPA recognize citrullinated proteins and come in different flavours;
• response broadens before disease manifestation disease and then stabilizes
• FceR+ cells from ACPA+ RA-patients are activated by citrullinated Antigens
• a direct functional response of immune cells from ACPA+ patients to citrullinated antigens
• Point to a role for IgE-ACPA and FceR+ cells in RA
Summary
Outline:
- Introduction; ACPA; IgE
- Homo-citrulline vs citrulline; Can ACPA discriminate?
- Anti-Carbamylated Protein Antibodies (anti-CarP
antibodies)
- Anti-CarP-antibodies in health and disease
Citrullination Carbamylation
Posttranslational modifications:
Citrulline and Homocitrulline
ACPA can discriminate between Citrulline
and Homocitrulline in the same peptide
Sequence of different forms
Arg NEE GFF SAR GHR PLD KK
Cit NEE GFF SACit GHR PLD KK
Homo NEE GFF SAHomoGHR PLD KK
Lys NEE GFF SAK GHR PLD KK
Dose dependent binding to Ca-FCS
Yet, antibodies recognizing carbamylated
proteins do exist
Anti-Carbamylated protein antibodies: anti-CarP
Shi et al. PNAS 2011
Anti-CarP antibodies and ACPA are
not cross-reactive
Shi et al. PNAS 2011
Anti-CarP-antibodies also bind to defined
carbamylated proteins
150
100
75
50
37
Ca-Fib Ci-Fib Fib Ca-Fib Ci-Fib Fib
ACPA-;
anti-CarP+
ACPA+;
anti-CarP-
Shi et al. ARD 2012
Anti-CarP antibodies and ACPA are
not necessarily cross reactive.
Start material (ACPA + Anti-CarP pos)
Flow
Through
ACPA neg
Anti-CarP pos
Eluate
ACPA +
cross reactive Ab
CCP column
Shi et al. ARD 2012
Outline:
- Introduction, ACPA; IgE
- Homo-citrulline vs citrulline; Can ACPA discriminate?
- Anti-Carbamylated Protein Antibodies (anti-CarP
antibodies)
- Anti-CarP-antibodies in health and disease
disease NHS RA UA
number 305 557 403
% positive 2.9% 44.9% 14.1%
NHS RA UA
305 557 408
5.2% 43.0% 19.9%
IgG IgA
Anti-CarP IgG and IgA are present in RA
Shi et al. PNAS 2011
Percentage positive sera in early RA for anti-
CarP-antibodies and ACPA
Percentage positive sera in early RA for anti-
CarP-antibodies and ACPA
Disease duration in years
SHARP score
1.5 5.0 12.5 20.0 21.5
23 56 96 113 123
Does the presence of Anti-CarP-antibodies predict progression of radiological damage in RA
Anti-CarP-antibodies predict radiological
damage in ACPA-negative disease
Are Anti-CarP-antibodies already present
pre-disease?
Collaboration with L. vd Stadt and D. van Schaardenburg
Are Anti-CarP-antibodies already present
pre-disease?
NHS arthralgia0
500
1000
1500
2000
2/32=6.3% 133/340=39,1%
An
ti-C
arP
Ig
G (
aU
/ml)
39%
Collaboration with L. vd Stadt and D. van Schaardenburg
NHS arthralgia0
500
1000
1500
2000
2/32=6.3% 133/340=39,1%
An
ti-C
arP
Ig
G (
aU
/ml)
39%
Anti-CarP pos
Anti-CarP neg
HR = 2.5
P<0.001
Anti-CarP antibody positive arthralgia patients
have an increased risk to develop RA
Collaboration with L. vd Stadt and D. van Schaardenburg
Shi et al. manuscript in preparation
Arthralgia cohort: ACPA and/or RF pos
Arthralgia to RA2010 increases with number of
autoantibodies present
Quiz: how much IgG molecules are
made by an ACPA producing B-
cell?
a) 500 per day
b) 500 per hour
c) 500 per minute
c) 500 per second
d) 500 per milli second
ACPA production speed of PB/PC
Average IgG Average
ACPA
Average
ACPA
ACPA of
IgG
IgG
producing B
cells
produce
ACPA
B
cells
/well
ACPA
produ
cing
Total
ACPA
produce
d
ACP
A
prod
uctio
n
ACPA
production
ng/ml U/ml ng/ml % 1 in x cells/
well
ng/well mole
cules
/seco
nd
molecules/
sec./ Bcell
PP2.
68 610 0.6 13 2.2% 46 520 11 1.32 87
62
779
Assumptions: All PB/PC produce IgG (therefore this number 779 per B cell is
an underestimation). ACPA producing B cells produce IgG (ACPA) with the
same rate as other B cells produce IgG. Culture FACSsorted PB/PC (out of
SFMC); Stimulated culture day 0-12, unstimulated culture after day 12.
Antibody production between day 12 and day 19
• Autoantibodies can discriminate between
citrullinated and homocitrulinated antigens
• Anti-CarP antibodies and ACPA do not cross react
• Anti-CarP antibodies are a new family of auto-
antibodies recognizing carbamylated proteins.
• Anti-CarP antibodies predict disease progression in
ACPA-negative disease and arthralgia patients.
• Anti-CarP antibodies may be a useful serological
marker to aid in diagnosing ACPA neg RA
Conclusion
ACPA
Anti-CarP
Acknowledgements
LUMC, Rheumatology:
Jing Shi
Priscilla Kerkman
Annemie Schuerwegh
Jolien Suurmond
Parawee Suwannalai
Rachel Knevel
Annette van der Helm
Diane van der Woude
Uli Scherer
Nivine Levarht
Leendert Trouw
Rene Toes
LUMC, Immunohematology
Peter van Veelen
George Janssen
The Dutch Arthritis Association
JBI/Reade Amsterdam
Lotte van der Stadt
Karin Britsemmer
Dirkjan van Schaardenburg
LUMC, Nephrology:
Tony Cerami