Autoantibody Assessment in Rheumatic Disease

Dana Ascherman, M.D.Division of Rheumatology

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

Raynaud’s-type vasospasm

slight fatigue, mild arthralgias

ROS otherwise negative

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

Case #1

Any additional physical exam maneuvers?

Should we order autoantibodies?

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

polyarthralgia/polyarthritis x 6 months

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

ROS otherwise negative

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

Labs: leukopenia, mild anemia, ESR=47

ANA positive 1:640 in speckled pattern

What tests to order next?

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

possible autoimmune disorder

Developed “pneumonia” in 2007—no response to antibiotics

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

functional, and radiologic improvement

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 #3Review 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 PresentationPhysical 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

mechanic’s hands, no muscle weakness

steroid-responsive ILD

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

What to do next (besides call a rheumatologist)?

Case #4 53 year old male presenting with polyarthritis,

intermittent fever, and weight loss

associated fatigue, sinus congestion, epistaxis

ROS otherwise negative except subjective hearing loss

Physical examination:• Gen: fatigued appearing• T=38.3• slight proptosis (right)• purpuric skin lesions distal lower extremities• mild synovitis PIPs, knees

Case #4

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

Imaging studies?

Additional serology?

Overview

1) Mechanisms of autoantibody formation

3) Disease associations

2) Methods of autoantibody detection

4) Autoantibodies as markers of clinical phenotype

Autoimmunity

“Horror Autoxicus”--Paul Ehrlich

Self-antigens targeted

Price of adaptive immunity

Question: why isn’t autoimmunity even more common?

Tolerance

Regulatory mechanism(s) to prevent uncontrolled autoreactivity

B vs. T cell tolerance

Central 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-γ

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

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

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

modifications may not occur in thymus--escape central tolerance

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

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

Antibody Detection Immunofluorescence

• ANA, ANCA

ELISA• CCP• U1RNP• SS-A/SS-B• Scl70

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

Immunodiffusion—tRNA synthetases

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- IIF 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

somewhat subjective

limited standardization of pattern reporting

MethodologyELISA

MethodologyELISA

Limitations- ELISApurity of Ag often a problem

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

antigenic proteins

some assays use only fragments of Ag

patients may have Ab to blocking agent

small operator inconsistencies can result in large errors

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

MethodologyImmunoprecipitation

Principlepatient’s Ab is bound to Protein

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

target antigen binds to the Ab

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

190

150

138

126

80

70

62

43

34

32

27

PL-12 110 kd Band

NormalRNA

PolymerasesKuSRP SclerodermaAbs

RNA Pol

KuBands

SRP 54 kd Band

SRPPL-12

Immunoprecipitation

ImmunoprecipitationLimitations

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

very large and very small antigens cannot be visualized

some antigens cannot be distinguished because they are of similar sizes

takes 1-2 weeks (batching)

MethodologyImmunodiffusion

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

at equivalence point, a visible preciptin line be formed

Autoantibody AssessmentOverall Limitations

RF, 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 TestingANA is useful to exclude lupus, not to

diagnose it

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

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

Autoantibodies:Markers of Clinical Phenotype

Myositis--Autoantibodies

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

Antibody Target Subset PhenotypeMi-2 NuRD DM Shawl, V-neck, Gottron’sCADM-140 MDA-5 DM Amyopathic, ILDSAE SUMO DM ILD, dysphagiaMJ NXP-2 JDM Calcinosis, Ulcerationp155/140 TIF1-γ DM, JDM Severe skin, malignancySRP 72, 54 kDa PM Severe/refractory myositisp200/100 HMGCR IMNM Necrotizing myopathyJo-1 ARS PM/DM Anti-synthetase syndrome

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

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

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

Any additional physical exam maneuvers?

Should we order autoantibodies?

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

1. Nailfold Capillaroscopy2. ANA

Case #2--Summary

fatigue, xerophthalmia, polyarthralgia, weakness, Raynaud’s

ANA positive 1:640 in speckled pattern

leukopenia, anemia, elevated ESR

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

Additional serology?

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

steroid-responsive ILD

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

Anti-Synthetase Syndrome (incomplete)—anti-EJ

Case #4--Summary

Serology?

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

anemia, Cr=2.3, hematuria, elevated ESR

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

Imaging?

CXR: pulmonary nodules

ConclusionAutoantibody formation reflects breakdown of tolerance

•combination of T and B cell dysregulation

Clinical picture should determine serological profiling

•RF, CCP—rheumatoid arthritis

Autoantibodies 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)

Caveat: RF, ANA lack specificity