Autoantibody Assessment in Rheumatic Disease

Dana Ascherman, M.D.Division of Rheumatology

Case #1Ø 19 year old female presenting with 2 year history of

Raynaud’s-type vasospasm

Ø slight fatigue, mild arthralgias

Ø ROS otherwise negative

Ø Physical examination:• no oral ulcerations• no skin rash, peri-ungual blush• no synovitis

Case #1

Ø Any additional physical exam maneuvers?

Ø Should we order autoantibodies?

Case #2Ø 37 year old female presenting with intermittent fever,

polyarthralgia/polyarthritis x 6 months

Ø slight fatigue, mild xerophthalmia, Raynaud’s, difficulty climbing stairs

Ø ROS otherwise negative

Ø Physical examination:• afebrile• slightly diminished tear pool• sclerodactyly; no skin rash• no synovitis, but multiple tender joints• 4+/5 deltoid, iliopsoas, quadriceps strength

Case #2

Ø Labs: leukopenia, mild anemia, ESR=47

Ø ANA positive 1:640 in speckled pattern

Ø What tests to order next?

Case #3Ø 43 year old male referred for evaluation of ILD and

possible autoimmune disorder

Ø Developed “pneumonia” in 2007—no response to antibiotics

Ø Diagnosis revised to “pulmonary fibrosis”• treated with prednisone for 1 year with clinical,

functional, and radiologic improvement

Ø Pulmonary evaluation: • restrictive PFTs with reduced FVC, TLC, DLCO• HRCT with basilar ground glass, minimal

honeycombing• Serology: -ANA, +SS-A, -Jo-1

Bilateral lower lobe air space disease with ground glass opacities consistent with active alveolitis; minimal fibrosis in lung bases peripherally

Case #3ØReview of Systems:

• no constitutional symptoms (F/C, NS, weight loss)• denies xerophthalmia/xerostomia, Raynaud’s• denies skin thickening or rashes beyond patchy

hyperpigmentation of palms• currently no cough, significant dyspnea• denies dysphagia, reflux, abdominal pain, change in

bowel habits• no joint pain/swelling/stiffness• mild proximal upper extremity aching without proximal

weakness

Case PresentationØPhysical Examination:

• afebrile, normal blood pressure, respiratory rate• grossly adequate tear, salivary pools• without palpable LAD• lungs with good air movement, no use of accessory

muscles; CTA without rales/rhonchi/wheezing• normal radial, posterior tibial pulses• musculoskeletal exam without synovitis• normal proximal, distal muscle strength• skin without Gottron’s rash/papules

Case #3Summary

Ø mechanic’s hands, no muscle weakness

Ø steroid-responsive ILD

Ø -ANA, -Jo-1, +SS-A cytoplasmic staining

What to do next (besides call a rheumatologist)?

Case #4

Ø 53 year old male presenting with polyarthritis,

intermittent fever, and weight loss

Ø associated fatigue, sinus congestion, epistaxis

Ø ROS otherwise negative except subjective hearing loss

Ø Physical examination:

• Gen: fatigued appearing

• T=38.3

• slight proptosis (right)

• purpuric skin lesions distal lower extremities

• mild synovitis PIPs, knees

Case #4

Ø Labs: Hgb=9.7, Cr=2.4, U/A: 1+ protein, 43 RBCs; ESR=83

Ø Imaging studies?

Ø Additional serology?

Overview

1) Mechanisms of autoantibody formation

3) Disease associations

2) Methods of autoantibody detection

4) Autoantibodies as markers of clinical phenotype

Autoimmunity

Ø“Horror Autoxicus”--Paul Ehrlich

ØSelf-antigens targeted

ØPrice of adaptive immunity

ØQuestion: why isn’t autoimmunity even more common?

Tolerance

ØRegulatory mechanism(s) to prevent uncontrolled autoreactivity

ØB vs. T cell tolerance

ØCentral vs. peripheral tolerance

Autoimmunity: Mechanisms

1) Molecular mimicry•Rheumatic fever, HSK, Guillain-Barre

2) Release of sequestered antigen•Trauma, infection

4) Upregulation of MHC Class II•Release of IFN-g

3) Generation of cryptic/neo-epitopes•Apoptosis--Granzyme B cleavage•?tissue-specific•Defective clearance--C1q deficiency (SLE)

5) Polyclonal B cell activation•EBV infection

6) Immune system defects, alterations•cytokine milieu--IBD

Antigen Processing/Presentation

Generation and Presentation of Autoantigens:Post-translational modifications

Ø influence Ag structure, immunogenicity–examples include deimination of arginine to citrulline (anti-CCP), deamidaiton of aspartic acid to isoaspartic acid, glycosylation, transglutamination, oxidative damage

Ømodifications can be triggered by aging, cellular stress induced by infection, trauma, apoptosis

Ømodifications may not occur in thymus--escape central tolerance

Øpost-translational changes could also influence subsequent processing, generation of epitopes

Ømay promote epitope spreading--B cell cross reactivity to modified, native version

Antibody DetectionØ Immunofluorescence

• ANA, ANCA

Ø ELISA• CCP• U1RNP• SS-A/SS-B• Scl70

Ø Immunoprecipitation• tRNA synthetases (e.g., Jo-1)• myositis- and scleroderma-specific autoantibodies

Ø Immunodiffusion—tRNA synthetases

Ø Nephelometry—RF

MethodologyImmunofluorescence

MethodologyImmunofluorescence

ANA--Patterns

PatternsA) Rim (dsDNA/chromatin) B) HomogenousC) Speckled (Ro/La, Sm/RNP) D) Nucleolar (Scl-70)

A) B)

C) D)

ACR slide collection

ANA Interpretation

ANCA

C-ANCA (PR3) P-ANCA (MPO)

Limitations- IIFØ antigen must be present in sufficient copy

number in the cell substrate • fixation solvent may strip antigen• cell may not produce sufficient quantity of Ag

Ø somewhat subjective

Ø limited standardization of pattern reporting

MethodologyELISA

MethodologyELISA

Limitations- ELISAØpurity of Ag often a problem

• recombinant Ag often expressed in bacteria• Ag purified from animal tissues may include other

antigenic proteins

Ø some assays use only fragments of Ag

Øpatients may have Ab to blocking agent

Ø small operator inconsistencies can result in large errors

ØAg coating on plates can be damaged by shipping and handling conditions

MethodologyImmunoprecipitation

PrincipleØpatient’s Ab is bound to Protein

A Sepharose-coated beads and then incubated with an extract derived from human cells

Ø target antigen binds to the Ab

Ø immunprecipitate is eluted, bound Ag is separated by electrophoresis and visualized

190150138

12680

7062

43343227

PL-12 110 kd Band

NormalRNA

PolymerasesKuSRP SclerodermaAbs

RNA Pol

KuBands

SRP 54 kd Band

SRPPL-12

Immunoprecipitation

ImmunoprecipitationLimitations

Ø proteins must be soluble, present in sufficient amount, and contain sufficient methionine for labeling

Ø very large and very small antigens cannot be visualized

Ø some antigens cannot be distinguished because they are of similar sizes

Ø takes 1-2 weeks (batching)

MethodologyImmunodiffusion

Øpatient sera and crude Ag mix are placed in wells in a gel an allowed to diffuse

Øat equivalence point, a visible preciptin line be formed

Autoantibody AssessmentOverall Limitations

ØRF, ANA lack specificity

SLE Present + -

+ 3 300

AN

A

Result - 1 9696

Lupus prevalence : ~ 4 in 10,000 in general populationProb of ANA >= 1:320 in SLE: ~ 75%

Prob of ANA >= 1:320 in normals: ~ 3%

Positive predictive value = 3/303 = ~1%Negative predictive value = 9696/9697 = >99.98%

Role of ANA TestingØANA is useful to exclude lupus, not to

diagnose it

Ø follow-up clinical assessment of ANA+ patients can be beneficial in high-risk populations

ØANA has no value in assessing prognosis or disease activity (i.e., do not follow titers)

Autoantibodies:Markers of Clinical Phenotype

Myositis--Autoantibodies

Ø target nuclear and cytoplasmic antigens: Mi-2, PM/Scl, SRP, Ku, tRNA synthetases

Antibody Target Subset Phenotype

Mi-2 NuRD DM Shawl, V-neck, Gottron’s

CADM-140 MDA-5 DM Amyopathic, ILD

SAE SUMO DM ILD, dysphagia

MJ NXP-2 JDM Calcinosis, Ulceration

p155/140 TIF1-g DM, JDM Severe skin, malignancy

SRP 72, 54 kDa PM Severe/refractory myositis

p200/100 HMGCR IMNM Necrotizing myopathy

Jo-1 ARS PM/DM Anti-synthetase syndrome

Myositis--AutoantibodiesJo-1 (histidyl-tRNA synthetase): 25%

Ø defines clinically homogeneous patient population: anti-synthetase syndrome (fever, myositis, arthritis, Raynaud’s, mechanic’s hands, ILD)

Ø range of clinical manifestations varies depending upon targeted sytnthetase autoantigen

Systemic Sclerosis:Antibody subsets and Clinical Phenotype

1) Anti-centromere: limited cutaneous, Raynaud’s, pulmonary HTN

2) Anti-Th/To: limited cutaneous, pulmonary HTN and/or ILD

3) Anti-toposisomerase I (Scl-70): diffuse cutaneous, GI, ILD, renal crisis

4) Anti-RNA polymerase III: diffuse cutaneous (rapid), renal crisis

5) Anti-PM/Scl: myositis/scleroderma overlap, ILD

Case #1

Ø Any additional physical exam maneuvers?

Ø Should we order autoantibodies?

Ø 19 year old female with mild arthralgia, +Raynaud’s

1. Nailfold Capillaroscopy2. ANA

Case #2--Summary

Ø fatigue, xerophthalmia, polyarthralgia, weakness, Raynaud’s

Ø ANA positive 1:640 in speckled pattern

Ø leukopenia, anemia, elevated ESR

ENA (SS-A, SS-B, Smith, RNP)--MCTD

Ø Additional serology?

Case #3--SummaryØ mechanic’s hands, no muscle weakness

Ø steroid-responsive ILD

Ø -ANA, -Jo-1, +SS-A cytoplasmic staining

Anti-Synthetase Syndrome (incomplete)—anti-EJ

Case #4--Summary

Ø Serology?

Ø 53 year old male with:• fever• weight loss, fatigue• epistaxis, hearing loss• polyarthritis

Ø anemia, Cr=2.3, hematuria, elevated ESR

ANCA (c-ANCA/PR3)—Granulomatosis with Polyangiitis

Ø Imaging?

Ø CXR: pulmonary nodules

ConclusionØAutoantibody formation reflects breakdown of tolerance

•combination of T and B cell dysregulation

ØClinical picture should determine serological profiling•RF, CCP—rheumatoid arthritis

ØAutoantibodies serve as biomarkers of disease subsets

•ANA—SLE (dsDNA, Sm), Sjogren’s (SS-A, SS-B, RF), MCTD (RNP), systemic sclerosis (Scl-70, RNApol III, centromere)

•cytoplasmic ANA/ANA negative--myositis

•ANCA—GPA (c-ANCA/PR3), mPAN, EGPA (p-ANCA/MPO)

ØCaveat: RF, ANA lack specificity