relevance of autoantibodies in ra - euro diagnostica wj huizinga... · tom huizinga malmo 2012 ....

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