SLIDE 1 Anticorps rares dans les myopathies inflammatoires et la sclérodermie
Camillo Ribi Médecin associé, MER Service d’Immunologie et Allergie
Formation continue IAL 6 novembre 2014
SLIDE 2 Dépistage auto-anticorps connectivites (LIA)
Recherche d’ANA (=FAN)
(Immunofluorescence indirecte sur lignée cellulaire Hep-2)
Titre 1/80 Négatif Titre 1/80 – 1/160 Ac anti-nucléosomes (ELISA)
Spécificités (ECL)
SSA, SSB, RNP, Sm, Scl-70, Jo1 Titre 1/320 Négatif Positif
- Ac. anti-nucléoprotéines (ECL)
Négatif Positif
Ac anti-dsDNA
homogène moucheté
SLIDE 3 Dépistage auto-anticorps connectivites (LIA)
Recherche d’ANA (=FAN)
(Immunofluorescence indirecte sur lignée cellulaire Hep-2)
Titre 1/80 Négatif Titre 1/80 – 1/160 Ac anti-nucléosomes (ELISA)
Spécificités (ECL)
SSA, SSB, RNP, Sm, Scl-70, Jo1 Titre 1/320 Négatif Positif
- Ac. anti-nucléoprotéines (ECL)
Négatif Positif
Ac anti-dsDNA
homogène centromère nucléolaire fluorescence cytoplasmique moucheté dot sclerodermie dot myosites
SLIDE 4
Myopathies inflammatoires Sclero-Poly-Synthétase Profile 12 dot Sclérose systémique EUROLINE Systemic Sclerosis Profile
Immuno-dot / line blot disponibles au LIA
SLIDE 5
Types of autoimmune inflammatory myopathies
anti- synthetases Immune-mediated necrotizing myositis
DM PM IBM CTD
SLIDE 6 Features of immune-mediated myopathies
adapté de Luo YB, Mastaglia FL. Biochim Biophys Acta 2014
SLIDE 7 Pathomechanisms in dermatomyositis
Ischemic myopathy
- deposition of immunoglobulins on intramuscular capillaries (?)*
- complement (C5b-9) mediated injury to endothelial cells
- depletion of the muscle capillary bed
- muscle fibre necrosis
- characteristic perifascicular pattern of muscle fibre atrophy
* unclear how the complement pathway is activated in DM auto-antibodies targeting endothelial cell antigens have never been identified Inflammatory cells in perimysial and perivascular infiltrates:
- B cells (upregulation of BAFF)
- CD4 + T helper cells (Th1 and Th17)
recruitement of mononuclear cells upregulation of MHC class I
- interferon-α (IFNα) producing plasmacytoid dendritic cells
SLIDE 8 Histological pattern of dermatomyositis
A. endomysial and perivascular mononuclear inflammatory infiltrate B. fragmentation of perimysial connective tissue and infiltration with granular mononuclear cells and macrophages C&D: perifascicular fibre regeneration and atrophy A–C: haematoxylin and eosin D: MHC-I immunohistochemistry
SLIDE 9
Antibodies in inflammatory myopathies
Various autoantibodies with high specificity Role in the pathogenesis of the muscle damage remains unclear Considerable racial and geographic differences in frequency Known to be influenced by genetic factors, particularly the HLA genotype Distinction of Myositis-specific Ab 30-58% of cases, mutually exclusive DM anti-Mi2, anti-TIF, anti-MDA5, anti-NXP2 Anti-synthétases anti-Jo1, anti-PL7, anti-PL12, anti-EJ Myosites nécrosantes anti-SRP, anti-HMGCR Myositis-associated Ab also found in other connective tissue diseases anti-SSA, anti-SSB, anti-U1RNP, anti-Ku, anti-PmScl...
SLIDE 10 Classic auto-antibodies in DM: Anti-Mi2
Target component of nucleosome remodelling deacetylase complex (NuRD) High levels of expression of Mi-2 in regenerating muscle cells and epidermis Some studies show correlation with level of UV light exposure
- > Role in sustaining the auto-immune process ?
Mainly in adult DM, rare in juvenile Reported frequency varies greatly (3–60%) Associated with:
- Typical DM skin changes
- Higher serum CK levels
- Good response to treatment
- Less likely to develop interstitial lung disease
- Less likely to develop cancer
SLIDE 11
Newer antibodies in DM: TIF-1 γ and TIF-1α
Antibodies to transcription intermediary factor 1 γ and α (TIF-1 γ, α) Target are 155/140 kDa nuclear proteins Indirect immunofluorescence: nuclear pattern Pathophysiological role of anti-TIF Ab not yet understood TIF generated as part of an anti-tumoral response ? Antibodies may cross-react with muscle/skin expressing high levels of TIF anti-TIF γ mostly coexist with anti-TIFα Both in juvenile and adult DM Positivity rate of 16–23% Adult patients have a higher incidence of malignancy
SLIDE 12
Newer antibodies in DM: anti-MDA5 (CADM-140)
Targets cytoplasmic RNA-specific helicase belonging to RIG-I-like receptor family Now known as ‘melanoma differentiation-associated gene 5’ (MDA5) MDA5 participates in the innate immune response to viruses Potential activator of type I IFN transcription First described in Japanese patients Considerable racial and ethnic variation in frequency and associated phenotype About 7% of DM (USA) usually: more severe skin manifestations (skin and oral ulcerations) higher incidence of rapidly progressive interstitial lung disease less muscle involvement (lower serum CK levels) picture sometimes resembling the anti-synthetase syndrome
SLIDE 13 MDA5: a cytosolic member of PRR
Bowie AG. Nature Reviews Immunology 2008
SLIDE 14 Clinical features of MDA5 + dermatomyositis
Fiorentino D. J Am Acad Dermatol 2011
SLIDE 15 Typical skin manifestations of anti-MDA5+ DM
Fiorentino D. J Am Acad Dermatol 2011
SLIDE 16
Newer antibodies in DM: Anti-NXP-2 (MJ, p-180)
Target: nuclear matrix protein 2 (NXP-2) Indirect immunofluorescence: nuclear Present in up to 25% of cases of juvenile DM Ethnic/geographic variability in frequency (Italy: 30% adult DM, Japan <2%) More common in patients of younger age Associated with More severe muscle weakness Calcinosis Joint contractures Intestinal vasculitis In adults: increased incidence of neoplasia, particularly in males
SLIDE 17
« Polymyositis »
SLIDE 18
« Polymyositis »
Most of the time part of a systemic autoimmune disease Mainly in adults, very rare in childhood Painless proximal myopathy with subacute onset Subvariants: Distal weakness mainly in the upper limbs, with good response to treatment Involvement of the paraspinal muscles with head drop (camptocormia) Usually responsive to corticosteroids and immunosuppression Pathogenesis: CD8 + T cell predominant lymphocytes invading non-necrotic fibres Interaction with antigen-presenting MHC-I molecules Doubt as to whether there is a common self-antigen Cytotoxicity trough release of perforin
SLIDE 19 Histological pattern of overlap-myositis
A–D: endomysial mononuclear inflammatory infiltrate myofibre invasion by mononuclear cells (C, arrowhead) myofibre necrosis (C, D); and diffuse MHC-I antigen expression (B).
A ,C: haematoxylin & eosin B: MHC-I immunohistochemistry D: modified Gomori trichrome.
SLIDE 20 Autoantibodies in PM
Anti-synthetase syndrome n.b. anti-Jo1 reported in up to 16% of DM Antibodies to synthetase are considered myositis-specific Mixed connective tissue disease (MCTD) high titres of antibodies to 70-kD ribonuclear protein complex (anti-U1 snRNP) features of systemic sclerosis / SLE such as Raynaud's phenomenon Sclerodactyly Synovitis Scleroderma–myositis overlap syndrome
- nly a small minority of cases of scleroderma
presence of antibodies to the nucleolar PM-Scl antigen complex (75 or 100kD)
SLIDE 21 Anti-synthetase syndrome
Characterized by antibodies to one of the tRNA synthetases (cytoplasma) Anti-Jo1 (anti-histidyl tRNA synthetase) most prevalent (20% of cases) Anti-Jo1 much more (5x) frequent than all the other anti-synthetase Ab Histopathologically: Perimysial connective tissue damage CD68 + histiocytes and perivascular CD4 + T cells perifascicular fibre necrosis and regeneration Clinically myositis + other systemic features, including
- interstitial lung disease (sometimes precedes myositis !)
- Raynaud's phenomenon
- deforming arthropathy
- skin changes in the hands
Condition is relatively resistant to treatment
SLIDE 22 Immune-mediated necrotizing myopathy
Heterogeneous group of acquired myopathies Distinguishable from DM and PM on clinical and pathological grounds Histopathologically: Myofibre necrosis Inflammation is usually lacking Immunostaining for C5b-9 and MHC-I in some cases Amenable to treatment with immune therapies May be classified into different categories, associated with
- malignancy
- connective tissue diseases
- viral infections (HIV, HCV)
- specific auto-antibodies: anti-SRP, anti-HMGCR and anti-synthetase
These antibodies are putatively involved in the pathogenesis...
SLIDE 23 Immune-mediated necrotizing myopathy
A,B: Associated with prolonged statin therapy; C,D: idiopathic case A: muscle fibre necrosis with sparse inflammatory infiltrate and B: diffuse MHC-I expression C: necrotising myopathy with D: scattered macrophagic infiltrate.
A, C: haemotoxylin & eosin B: MHC-I immunohistochemistry D: CD163 immunohistochemistry
SLIDE 24 Myopathy associated with anti-SRP antibody
Anti-SRP previously considered a specific antibodies associated with PM or DM Myopathy associated with anti-SRP antibody now stands as a separate entity Infrequently associated with other autoimmune diseases Clinically: rapidly progressive > chronic proximal muscle weakness high serum CK levels relative resistance to treatment with conventional immune therapies Histopathologically:
- active necrotising myopathy
- inconspicuous or absent inflammatory infiltrate and MCH-I up-regulation
- occasionnally C5b-9 deposition on endomysial capillaries and muscle fibres
- predominance of macrophages
SLIDE 25 Myopathy associated with anti-SRP antibody
Signal recognition protein = cytoplasmic ribonucleoprotein complex Transfers newly synthesised peptides from ribosomes to the ER Not clear how anti-SRP antibody lead to muscle fibre necrosis (SRP is ubiquitous) But:
- Correlation between serum levels of the antibody and CK levels
- Response to plasma exchanges
- Increasing evidence for ADCC
Favours a causal role for the antibody
SLIDE 26 Statin myotoxicity
Statins = 3-OH-3-methyl-glutaryl-CoA reductase (HMGCR) inhibitors) Muscular injury ranges from simple myalgia to severe necrotising myopathy Risk of myopathy varies with the different statins Several pathomechanisms have been proposed Predisposing factors include
- age
- female gender
- other co-morbidities
- concomitant medications
- genetic predisposition
majority of cases of statin myopathy are self-limiting progressive improvement of muscle symptoms and normalisation of CK levels
SLIDE 27 Anti-HMGCR associated Necrotizing Myositis
Small subgroup of patients who develop an IMNM:
- symptoms of muscular injury continue to progress
- may even first develop after the statin is withdrawn
In these patients, an autoimmune process is initiated:
- presence of a highly specific anti-HMGCR antibody
- not found in patients with other forms of statin myopathy
- antibody may also be found in some patients without prior statin exposure
- antibody levels correlate with serum CK levels as well as upper limb strength
Expression of HMGCR is up-regulated in regenerating muscle fibres Persistent over-expression of autoantigen may sustains autoimmune process Serum has to be sent to: Pr Olivier Boyer, CHU de Rouen
SLIDE 28 Anti-PmScl in systemic sclerosis with ILD
Guillen-Del Castillo A. Semin Arthritis Rheum 2014
SLIDE 29 Autoantibodies in systemic sclerosis
Important part of initial assessment in patients with suspected SSc
- diagnostic value
- risk prediction
Autoantibody specificities do not change over time Antibody levels have no clear relationship with disease activity and severity For anti-Scl70 Ab this paradigm is probably false:
- levels of anti-Scl70 Ab may correlate with the degree of fibrosis
Hu PQ. Arthritis Rheum 2003
n.b.: this is also true for
- anti-Jo1 antibodies associated with disease activity (muscle, joint, lung)
Stone KB. Arthritis Rheum 2007
- > Could assessment of anti-Jo1/anti-Scl70 be of use for follow-up ?
Opportunity to have now electrochemiluminescence for ENA specificities
SLIDE 30
Sclérose systémique: deux formes distinctes
Anticorps anti-centromères Anticorps anti-Scl70 atteinte cutanée diffuse (dcSSc) atteinte cutanée limitée (lcSSc) RNA-Pol III
SLIDE 31
Atteinte d’organes selon sous-type
Temps (années) Atteinte viscérale 1 2 3 Rein Poumon Coeur Oesophage Intestin HTAP lcSSc dcSSc Tube digestif
SLIDE 32 Classification criteria for systemic sclerosis
Van den Hoogen F. Ann Rheum Dis 2013
The American College of Rheumatology/European League Against Rheumatism criteria for the classification of systemic sclerosis Item Sub-item(s) Weight/score† Skin thickening of the fingers of both hands extending proximal to the metacarpophalangeal joints (sufficient criterion) – 9 Puffy fingers 2 Sclerodactyly of the fingers * 4 Fingertip lesions (only count the higher score) Digital tip ulcers 2 Fingertip pitting scars 3 Telangiectasia – 2 Abnormal nailfold capillaries – 2 Pulmonary arterial hypertension 2 Interstitial lung disease 2 Raynaud's phenomenon – 3 SSc-related autoantibodies (maximum score is 3) Anticentromere Anti–topoisomerase I Anti–RNA polymerase III *distal to the metacarpophalangeal joints but proximal to the proximal interphalangeal joints Not applicable to patients with skin thickening sparing the fingers or to patients who have a scleroderma-like disorder that better explains their manifestations (eg, nephrogenic sclerosing fibrosis, generalised morphea, eosinophilic fasciitis, scleredema diabeticorum, scleromyxedema, erythromyalgia, porphyria, lichen sclerosis, graft-versus-host disease, diabetic cheiroarthropathy). †The total score is determined by adding the maximum weight (score) in each category. Patients witha total score of ?9 are classified as having definite systemic sclerosis. 3 Skin thickening of the fingers (only count the higher score) Pulmonary arterial hypertension and/or interstitial lung disease (maximum score is 2)
SLIDE 33 Autoantibodies in systemic sclerosis
Domsic R. Curr Opin Rheumatol 2014
SLIDE 34 Autoantibodies in systemic sclerosis
Domsic R. Curr Opin Rheumatol 2014
SLIDE 35 Take-home messages
Auto-antibodies in 60-80 % of patients with inflammatory myopathies/sSclerosis Search for rare antibodies by dot if:
- suspected inflammatory myopathy
- changes on hands and fingers (dermatomyositis/mechanics hands)
- secondary raynaud’s (abnormal capillary pattern) or puffy fingers
- interstitial lung disease
Best if cytoplasmic fluorescence at ANA screen / ANA pos with nucleolar pattern Antibody specificites may be linked to particular features: anti-MDA5 and anti-synthetase antibodies severe ILD anti-TIF, NXP-2 (adults) cancer NXP-2 calcinosis anti-RNA-polymerase III scleroderma renal crisis
