Relevance of Autoantibodies in RA

Transcription

1 Relevance of Autoantibodies in RA Tom Huizinga Malmo 2012

2 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%

3 Amount of ACPA producing B cells ACPA production by unstimulated PBMC s Average IgG Average ACPA Average ACPA ACPA of IgG IgG producing B cells produce ACPA PP2.60 PP2.61 PP2.63 PP2.66 ng/ml U/ml ng/ml % 1 in x % % % % 255 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

4 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%

5 Amount of ACPA producing B cells ACPA production by unstimulated SFMC s 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 PP2.69 PP % % % 8

6 Outline: - Antibodies to post translational modifications - ACPA epitope recognition profile (Fine-specificity) - Relevance of isotypes? - Quantification per cell per time-unit

7 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

8 Arthritis Autoantibodies and immune complexes Chronic Inflammation and bone erosions

9 Anti-Citrullinated Protein Antibodies (ACPA) - 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

10 Differences in ACPA pos vs ACPA neg RA ACPA positive ACPA negative Genetics Environment Progression Remission Severity ACPA + RA ACPA - RA

11 Differences in ACPA pos vs ACPA neg RA ACPA positive ACPA negative Genetics Environment Progression Remission Severity ACPA + RA ACPA - RA

12 Anti-Citrullinated Protein Antibodies (ACPA) recognize various citrullinated substrates Anti-MCV Anti-CCP-2 Anti-CCP-3 Citr. Fibrinogen Citr. Fibronectin Citr. Vimentin Citr. MBP Citr. Enolase Citr. Collagen Et cetera

13 Citr. Fibr fibr Citr. Fibr fibr Citr. MBP MBP Citr. MBP MBP Anti-CCP-antibodies are ACPA and recognize different citrullinated proteins ACPA-positive serum REACTIVITY TO CITR. MBP Serum Eluate CCP column REACTIVITY TO CITR. FIBRINOGEN Serum Eluate

14 Extensive cross-reactivity of ACPA towards different citrullinated proteins Isolation of anti-cit-fibrinogen antibodies Isolation of anti-cit-mbp antibodies Ioan-Facsinay et al. Ann Rheum Dis 2011

15 ACPA-fine-specificity Vimentin peptide A Vimentin peptide B Fibrinogen peptide A Fibrinogen peptide B Enolase peptide

16 ACPA-fine-specificity and subgrouping of patients (using 8 peptides) cvim1-16 cvim59-74 cfib α cfib β ceno5-20 cmbp MCV CCP3 53 subgroups Willemze et al. Ann Rheum Dis 2011

17 Epitope recognition does not associate with clinical characteristics Willemze et al. Ann Rheum Dis 2011

18 Higher anti-ccp-2 levels associate with enhanced epitope recognition CCP2 level RF ANF cvim1-16 cfibα ceno5-20 cvim59-74 cmbp cfibβ CCP3 MCV CCP2level >1000 AU CCP2level=25 AU

19 Biology of anti-citrulline immune responses; fine-specificity 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 RF ANF cvim1-16 cfibα ceno5-20 cvim59-74 cmbp cfibβ CCP3 MCV Epitope recognition is correlated with ACPA levels Willemze et al, ARD 2012 CCP2 level epitope spreading before disease onset, then stable vd Woude et al, ARD 2010

20 Biology of anti-citrulline immune responses; isotype-usage and avidity Avidity of ACPA is low as compared to anti-recall responses Suwannalai et al, ARD 2011; Suwannalai et al., A&R 2011 IgG 1 99% IgG 2 82% IgG 3 60% IgG 4 98% IgM 61% IgA 62% IgE 80% 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

21 A broader isotype usage is associated with Radiographic progression EAC-Netherlands EURIDISS-Norway * comparison 4 isotypes versus 5 isotypes: p<0.05

22 IgE is associated with immunity against parasitic infections

23 The presence of IgE to allergens is classically measured by a Basophil Activation Test Ag IgE IgE CD63 20 min 37 C On ice Anti-CD63

24 Are FceRI-positive cells from ACPA-positive patients activated by citrullinated antigens? Allergen IgE Fc epsilon RI Citr. Ag IgE Fc epsilon RI FcεRI positive cell (mast cell/ basophil)

25 Direct activation of IgE-positive basophils by citrullinated Antigens? Citr. Ag IgE Anti-IgE CD63 20 min 37 C On ice Anti-CD63 Schuerwegh et al. PNAS 2010

26 % of basophils with CD63 basophil activation upregulation basophil activation Direct activation of IgE-positive basophils by citrullinated Antigens ACPA-positive RA ACPA-negative RA aige PAD NC-FB C-FB 0 - aige PAD NC-FB C-FB Schuerwegh et al. PNAS 2010

27 Mast cells exposed to ACPA+ RA serum are activated by Citrullinated proteins 10 4 Ctr aige Fib Citr. Fib Ctr 10 4 aige Fib Cit-Fib CD203c-APC CD203c-APC CD203c-APC CD203c-APC CD63-PE CD63-PE CD63-PE CD63-PE % CD63+ mast cells (delta) Degranulation * * IL-8 (ng/ml) IL-8 Ctr aige Fib Cit-Fib Ctr aige Fib Cit-Fib November 24,

28 Increased number of degranulated mast cells in synovial tissue of ACPA+ RA-patients All patients N=50 ACPA positive N=27 ACPA negative N=23 p-value CD117 + Mast Cells (MC) degranulated MC CD117+ CAE- 27 (0-178) 33 (0-178) 11 (0-100) (0-173) 28 (0-173) 6 (0-67) % degranulated MC 82 (0-100) 89 (0-100) 58 (0-100) 0.03 Schuerwegh et al. PNAS 2010

29 Elevated IgE expression on ACPA+ Mast Cells 2500 p< p= D MFL IgE expression OA ACPA - RA ACPA + RA Schuerwegh et al. PNAS 2010

30 Summary 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

31 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

32 Posttranslational modifications: Citrulline and Homocitrulline Citrullination Carbamylation

33 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

34 Yet, antibodies recognizing carbamylated proteins do exist Dose dependent binding to Ca-FCS Anti-Carbamylated protein antibodies: anti-carp Shi et al. PNAS 2011

35 Anti-CarP antibodies and ACPA are not cross-reactive Shi et al. PNAS 2011

36 Anti-CarP-antibodies also bind to defined carbamylated proteins Ca-Fib Ci-Fib Fib Ca-Fib Ci-Fib Fib ACPA-; anti-carp+ ACPA+; anti-carp- Shi et al. ARD 2012

37 Anti-CarP antibodies and ACPA are not necessarily cross reactive. Start material (ACPA + Anti-CarP pos) CCP column Flow Through ACPA neg Anti-CarP pos Eluate ACPA + cross reactive Ab Shi et al. ARD 2012

38 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

39 Anti-CarP IgG and IgA are present in RA IgG IgA disease NHS RA UA number % positive 2.9% 44.9% 14.1% NHS RA UA % 43.0% 19.9% Shi et al. PNAS 2011

40 Percentage positive sera in early RA for anti- CarP-antibodies and ACPA

41 Percentage positive sera in early RA for anti- CarP-antibodies and ACPA

42 Does the presence of Anti-CarP-antibodies predict progression of radiological damage in RA Disease duration in years SHARP score

43 Anti-CarP-antibodies predict radiological damage in ACPA-negative disease

44 Are Anti-CarP-antibodies already present pre-disease? Collaboration with L. vd Stadt and D. van Schaardenburg

45 Anti-CarP IgG (au/ml) Are Anti-CarP-antibodies already present pre-disease? % 0 NHS arthralgia 2/32=6.3% 133/340=39,1% Collaboration with L. vd Stadt and D. van Schaardenburg

46 Anti-CarP IgG (au/ml) Anti-CarP antibody positive arthralgia patients have an increased risk to develop RA 2000 Anti-CarP pos % Anti-CarP neg NHS arthralgia 2/32=6.3% 133/340=39,1% HR = 2.5 P<0.001 Arthralgia cohort: ACPA and/or RF pos Shi et al. manuscript in preparation Collaboration with L. vd Stadt and D. van Schaardenburg

47 Arthralgia to RA2010 increases with number of autoantibodies present

48 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

49 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 ng/ml U/ml ng/ml % 1 in x cells/ well Total ACPA produce d ng/well PP % ACP A prod uctio n mole cules /seco nd ACPA production molecules/ sec./ Bcell 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

50 Conclusion Autoantibodies can discriminate between citrullinated and homocitrulinated antigens Anti-CarP antibodies and ACPA do not cross react ACPA Anti-CarP antibodies are a new family of autoantibodies recognizing carbamylated proteins. Anti-CarP antibodies predict disease progression in ACPA-negative disease and arthralgia patients. Anti-CarP Anti-CarP antibodies may be a useful serological marker to aid in diagnosing ACPA neg RA

51 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 JBI/Reade Amsterdam Lotte van der Stadt Karin Britsemmer Dirkjan van Schaardenburg LUMC, Nephrology: Tony Cerami The Dutch Arthritis Association