Serological Markers

in Rheumatoid Arthritis

Rheumatoid Arthritis

Systemic autoimmune disease

Characterized by chronic inflammation of the joints resulting in tissue degradation and joint destruction

1-2% of the world population

Diagnosis – primarily on clinical manifestation, with only limited serological support

Clinical Manifestation of RA

1. Morning stiffness2. Arthritis of 3 or more joint areas3. Arthritis of hand joints4. Symmetric arthritis5. Rheumatoid nodule6. Serum rheumatoid factor7. Radiographic changeAt least 4 of these criteria(1-4 at least for

6 wks)

Radiological Manifestations of RA

Periarticular soft tissue swellingOsteoporosisMarginal erosions progressing to severe e

rosions of subchondral boneSynovial cyst formation and lack of bone r

epairHand, feet, knee, hips, cervical spine, sho

ulder, elbow

Association of RA with HLA

HLA-DR4, sometimes DR1associated with RA, but association is variable.

Functional similarity : Shared epitope a.a. sequence in the HVR3 of the chain (QK/

RRAA at positions 70-74 of the HLA DRB1 chain)

Association of HLA with RA in KoreansHong, et al. J Rheumatol 1996 DR4 61% vs 29% RR=3.7 Shared epitope 57% vs 22% RR=4.8 DRB1*0405 RR=9.4 DRB1*0401 RR=8.8

Kim, et al. Tissue Antigens 1999 DRB1*0405 RR=6.6 DQA1*03 RR=5.2 DQB1*04 RR=3.5 DQCAR113 RR=3.2 DQCAR115 RR=3.6 DQCAR 113/115 RR=11.2

Serological Markers in RA1. RA factor (IgM) IgG/ IgA2. Filaggrin associated Abs 1) antiperinuclear factor 2) antikeratin Ab 3) anticitrullinated filaggrin Ab3. Anti-RA 334. Anti-Sa5. pANCA6. Other autoAb: HSP 60, type II collagen, calretic

ulin, calpastain, MTOC(?), GiM(?)

Rheumatoid Factors (RF)

Ab to directed to the constant region of IgG

Usually IgM type, but IgG, IgA

Agglutination, nephelometry, ELISA based test

Detected in 70-80% of RA, in high % in other autoimmune and infectious diseases, in up to 15% of healthy individuals

Anti-perinuclear Ab(1)

Described by Nienhuis and Mandema, in1964

Ab to a component of a number of so-called keratohyaline granules surrounding the nucleus in IIF using buccal mucosa cells

Profilaggrin, precursor of filaggrin colocalize with PF

Keratohyalin granule of epidermis contains profilaggrin.

Sensitivity 49-91%, specificity 73-99%

APF positive buccal mucosa donor ~5%

Correlated with bone erosion in short term

Anti-perinuclear Ab(2)

Anti-keratin Ab(1)

Described by Young in 1979

Determined by IIF on cryosection of rat esophagus – stratum corneum of rat eosophagus epithelium

High correlation with APF

Not cytokeratin

A neutral/acidic variant of human epidermis filaggrin, a terminal differentiation protein involeved in the aggregation of cytokeratin filament during cornification

Sensitivity 36-59%, Specificity 88-99%

Anti-keratin Ab(2)

Anti-filaggrin Ab

Filaggrin, cytokeratin filament aggregating protein – the target of the so called antikeratin antibodies (Simon et al., 1993, J Clin Invest)

APF, AKA, anti-filaggrin extracted from human epidermis have similar diagnostic value… partially overlap, not totally identical (Vincent et al., 1999, Ann Rhem Dis)

APF AKA AFA RA % Control %N N N 71 25.4 186 87.3

P 11 3.9 4 1.9P 7 2.5 9 4.2

P 19 6.8 8 3.8P P 3 1.1 0 0

P P 11 3.9 1 0.5P P 38 13.6 3 1.4P P P 119 42.7 2 0.9

279 213

Distribution fo RA and control serum samples with regard to AKA, AFA and APF positivity

Vincent, et al. Ann Rheum Dis 1999

AFA immunoblot on filaggrin enriched epidermis extract

RF Filaggrin associated AbAPF AKA(AF

A)AFA

Sensitivity

(%)

70-80 40-80 35-55 Similar to APF/AKA

Specificity

(%)

80-90 >90 >90 Similar to APF/AKA

Good

Good ass /w prognosis

Specific, Appear early, Less change of titer

Stable Ag, ELISA or IB methods

Bad

Seronegative RF, Appear late, Titer fluctuation

Less correl/wprognosisLee available substrate,IF method

IF method, Less sensitive than APF

Less sensitive than APF

Comparison of Serological Markers in RA

Anti-Citrullinated Filaggrin Ab

RA associated Ab to filaggrin label the fibrous matrix of cornified cells but not the profillagrin containing keratohyalin granules in human epidermis (Simon et al., 1995, Clin Exp Immunol)

RA specific autoAb recognize citrulline, posttranslational modified arginine residue (Simon et al., 1998, J Clin Invest)

Arginine

Profilaggrin: an acidic phosphorylated protein, Consisting of 10 –12 tandemly repeated filaggrinunits separated by linker peptides

DephophorylatedProteolytically cleaved

Filaggrin: basic/neutral intermediate filament associated protein

Citrulline

Citrullination bypeptidyl arginine deaminase

Citrullinated Filaggrin

Cornification

Epidermis

Simon et al., 1998, J Clin Invest

Simon et al., 1998, J Clin Invest

RAN number Positive %

RF+ RA 34 21 61.76Prev RF+/Present RF- RA 9 7 77.78RF- RA 10 6 60.00Total 53 34 64.15

Control groupN number Positive %

RF+ non- RA 12 1 8.33RF- healthy donor 22 1 4.55Total 34 2 5.88

Anti-citrullinated Filaggrin Antibody on ELISA(MBL)

Sensitivity 64%, Specificity 94%, Efficiency 76%

87 sera tested on Hallym University Medical Center

Specific presence of intracellular citrullinated proteins in rheumatoid arthritis synovium: Relevance to anti

filaggrin autoantibodiesBaeten et al., Arthritis Rheum 2001

Objectives: investigate the presence of citrullinated proteins in synovial membrane of RA Pt

Methods: stain synovial tissue section of 88 RA pts and 52 other control pt with anti citrulline polyAbs(ACA) and affinity purified antifilaggrin autoantibody(AFA)

Results: intracellularly staining of the lining and sublining layers of RA synovial tissue with ACA and AFA, which were colocalized

Prognostic factors in early RA

Important clinical outcomes joint damage and functional disabilityPrognostic factors Joint involvement, RF positivity, ESR & CRP IgA RF, RF + AKA or AFA : more active Genetic predictors over RF : inconclusiveGuideline for aggressive treatment

Juvenile RAChronic inflammatory disease of unknown etiology and p

athogenesis1. Polyarticular onset JRA (=>5 joints) 30% 1) younger children RF(LA) negative 2) older girl RF(LA) positive

2. Pauciarticular onset JRA (1-4 joints) 50% 1) early onset, young girl /w iridocyclitis, positive ANA 2) late onset, boy /w HLA B27, ankylosing spondylitis

3. Systemic onset JRA ( fever, arthritis, skin rash, etc) 20%

Serological Markers for Juvenile RA(1)

1. RF RF_LA 7-10% JRA, late onset polyarticular ELISA IgM RF 22-35% IgG RF 4-6% IgA RF 30-60% Hidden IgM RF not detected by conventional methods using IgM containing fraction from sephadex chromatography 65(systemic onset) – 85% (polyarticular)

Serological Markers for Juvenile RA(2)

2. ANA 50-70% positive in JRA young girl with pauciarticular onset, iridocyclitis 95-100% positive ANA positivity well known risk factor for development of ant. uveitis Speckled and homogeneous No association with Sm, RNP, Ro, La, Scl70 Ab to Histone 40-70% of JRA

Serological Markers for Juvenile RA(3)

3. Other antibodies anticardiolipin antibody 7-42% anticollagen antibody 12-42% antiperinuclear factor non – 40%