MICHELLE KAHLENBERGSENIOR TALK

MARCH 19TH/20TH 2009

Understanding LupusOR

Why Rheumatology Is So Cool.

Why is lupus such a mystery to us?

Unlike many other autoimmune diseases, it affects many organs with varying disease manifestations over time.

This makes it difficult to diagnose: average is 4 years and 3 different doctors

There are all those antibodies to know…Treatments are OLD with significant side

effects

Talk Overview

Discuss Lupus diagnostic criteria and clinical manifestations

Explore Mechanisms of Lupus (cool!) Defective apoptosis IFN- TLR signaling B-cell disregulation Pathology of autoantibodies

Discuss how research into understanding the mechanisms of SLE is advancing treatment for lupus

Lupus Diagnostic Criteria (need 4)

1. Malar Rash: Fixed erythema, flat or raised, over the malar eminences, tending to spare the nasolabial folds

2. Discoid rash: Erythematous raised patches with adherent keratotic scaling and follicular plugging; atrophic scarring may occur in older lesions

3. Photosensitivity: Skin rash as a result of unusual reaction to sunlight, by patient history or physician observation

4. Oral ulcers: Oral or nasopharyngeal ulceration, usually painless, observed by physician

From http://www.rheumatology.org/publications/classification/SLE/1997UpdateOf1982RevisedCriteriaClassificationSLE.asp?aud=pat

Lupus Diagnostic Criteria (need 4)

5. Nonerosive Arthritis: Involving 2 or more peripheral joints, characterized by tenderness, swelling, or effusion

6. Pleuritis or Pericarditis: a) Pleuritis--convincing history of pleuritic pain or rubbing heard by a physician or evidence of pleural effusion OR b) Pericarditis--documented by electrocardigram or rub or evidence of pericardial effusion

7. Renal Disorder: a) Persistent proteinuria > 0.5 grams per day or > than 3+ if quantitation not performed OR b) Cellular casts--may be red cell, hemoglobin, granular, tubular, or mixed

8. Neurologic Disorder: a) Seizures--in the absence of offending drugs or known metabolic derangements; e.g., uremia, ketoacidosis, or electrolyte imbalance OR b) Psychosis--in the absence of offending drugs or known metabolic derangements, e.g., uremia, ketoacidosis, or electrolyte imbalance

9. Hematologic Disorder: a) Hemolytic anemia--with reticulocytosis OR b) Leukopenia--< 4,000/mm3 on ≥ 2 occasions OR c) Lymphopenia--< 1,500/ mm3 on ≥ 2 occasions OR d) Thrombocytopenia--<100,000/ mm3 in the absence of offending drugs

10. Immunologic Disorder: a) Anti-DNA: antibody to native DNA in abnormal titer OR b) Anti-Sm: presence of antibody to Sm nuclear antigen OR c) Positive finding of antiphospholipid antibodies on: an abnormal serum level of IgG or IgM anticardiolipin antibodies, a positive test result for lupus anticoagulant using a standard method, or a false-positive test result for at least 6 months confirmed by Treponema pallidum immobilization or fluorescent treponemal antibody absorption test

11. Positive Antinuclear Antibody: An abnormal titer of antinuclear antibody by immunofluorescence or an equivalent assay at any point in time and in the absence of drugs  

From http://www.rheumatology.org/publications/classification/SLE/1997UpdateOf1982RevisedCriteriaClassificationSLE.asp?aud=pat

Arthritis

Painful joints are #1 symptom in 76-100% of patients With or without signs of inflammation, mild effusions

if any Usually involve hands, wrists, knees Can be asymmetric Generalized myalgias particularly deltoids and quads

<15% with elevations in CPK

Nephritis

Very closely linked with prognosis 5- and 10-year survival rates are documented as high as

85% and 73%, respectively (used to be about 0% before immunosuppressants)

Diagnosed by presence of proteinuria (>500mg/24 hours) Watch for symptoms of edema, puffy eyes, frequent

urination.

Biopsy helpful to determine whether aggressive treatment is required

Associated HTN needs aggressive Rx with ACE/ARB

Nephritis

Class I Minimal mesangial lupus nephritis Normal glomeruli by light microscopy, but mesangial immune deposits by immunofluorescence

Class II Mesangial proliferative lupus nephritis Purely mesangial hypercellularity of any degree or mesangial matrix expansion by lightmicroscopy, with mesangial immune deposits May be a few isolated subepithelial or subendothelial deposits visible by immunofluorescence or electron microscopy, but not by light

microscopy Class III Focal lupus nephritisa

Active or inactive focal, segmental or global endo- or extracapillary glomerulonephritis involving 50% of all glomeruli, typically with focal subendothelial immune deposits, with or without mesangial alterations Class III (A) Active lesions: focal proliferative lupus nephritis Class III (A/C) Active and chronic lesions: focal proliferative and sclerosing lupus nephritis Class III (C) Chronic inactive lesions with glomerular scars: focal sclerosing lupus nephritis

Class IV Diffuse lupus nephritisb

Active or inactive diffuse, segmental or global endo- or extracapillary glomerulonephritis involving 50% of all glomeruli, typically with diffuse subendothelial immune deposits, with or without mesangial alterations. This class is divided into diffuse segmental(IV-S) lupus nephritis when 50% of the involved glomeruli have segmental lesions, and diffuse global (IV-G) lupus nephritis when 50% of the involved glomeruli have global lesions. Segmental is defined as a glomerular lesion that involves less than half of the glomerular tuft. This class includes cases with diffuse wire loop deposits but with little or no glomerular proliferation Class IV-S (A) Active lesions: diffuse segmental proliferative lupus nephritis Class IV-G (A) Active lesions: diffuse global proliferative lupus nephritis Class IV-S (A/C) Active and chronic lesions: diffuse segmental proliferative and sclerosing lupus nephritis Active and chronic lesions: diffuse global proliferative and sclerosing lupus nephritis Class IV-S (C) Chronic inactive lesions with scars: diffuse segmental sclerosing lupus nephritis Class IV-G (C) Chronic inactive lesions with scars: diffuse global sclerosing lupus nephritis

Class V Membranous lupus nephritis Global or segmental subepithelial immune deposits or their morphologic sequelae by light microscopy and by immunofluorescence or

electron microscopy, with or without mesangial alterations Class V lupus nephritis may occur in combination with class III or IV in which case both will be diagnosed

Class VI Advanced sclerosis lupus nephritis 90% of glomeruli globally sclerosed without residual activity

Table from Weening et al., J Am Soc Nephrol 15: 241–250, 2004

Nephritis

Class I-no RxClass II-Rx if proteinuria >1000 mg/dClass III and IV at high risk of progression so

require aggressive immunosuppressive therapy

Class V Rx with steroidsClass VI-dialysis or transplant

Nervous System

Headache is the most common complaintADD, mood disorders, anxiety, delirium,

psychosis, seizures (generalized or partial) Difficult to prove absolute causality

Generalized encephalopathies Formal neuropsychiatric testing reveals deficits in 21-67%

of patients with SLECerebritis

Degenerative changes in small vessel walls, often with minimal or no inflammatory infiltrates

May be related to immune complex depositionNeuropathy secondary to vasculitis of vasa

nervorum (often with dermatomyositis overlap)

CV System

Pericarditis 6-45% of patients: low likelihood of tamponade or constrictive type.

<10% with myocarditis Libman-Sacks endocarditis

1-4 mm vegetations of accumulations of immune complexes and mononuclear cells on mitral, tricuspid or aortic valves Risk of thromboembolism or secondary infective endocarditis (abx prophy)

Aortic insufficiency is the most common valvular abnormality. Heart disease

Contributes to bimodal pattern of mortality from lupus A study from U of Pittsburgh comparing rates of MI to that of Framingham Offspring

Study data showed that risk of MI was 50x higher in woman with lupus ages 35-44 and 2.5-4x higher in older age groups

Autopsy data shows CAD in 40% of SLE patients as opposed to only 2% of age matched controls.

Atherosclerotic plaque burden (via carotid intima media thickness measurements and by coronary calcium scores) is higher in patients with SLE than in controls

“Lupus dyslipoproteinemia” is low HDL, high TG, normal or only slightly elevated LDL, increased lipoprotein(a): this appears to correlate with disease activity

Means of prevention focus on risk factor management and inflammation control, but no clear guidelines are available as of yet.

Lungs and Pleura

Over 30% will have pleuritis or an effusion over the course of their disease Fluid is exudative, normal glucose, high protein, WBC

<10,000 (neutrophilic or lymphocytic), decreased complement

Can have pneumonitis, pulmonary hemorrhage (rare but often fatal), PE, pulmonary HTN Pulmonary HTN more likely to be associated with

Raynaud’s

Hematologic System

Chronic anemia is present in up to 80% of patients

Leukopenia is present in up to 50% of patients (lymphopenia more common than neutropenia).

Thrombocytopenia ranges from modest to severe with bleeding complications May reflect disease activity May be first sign of SLE; predating other signs and

symptoms by years. Associated with the presence of anti-platelet antibodies

Secondary APS seen in about 40% of patients with SLE

BUT WHAT CAUSES ALL OF THESE SYMPTOMS?

B cell differentiation:-negative selection of autoreactive cells in bone marrow (90%)-migration to LN-somatic hypermutation-survival by positive selection -requires numerous co-stimulatory interactions with APC and T cells (including upregulation of BAFF and IFN-)

Image from Niiro and Clark, Nature Reviews Immunology 2, 945 - 956

Defective clearance of apoptotic cells

One common theme is defects in clearance of apoptotic cells resulting in autoantibody production

Phagocytes from lupus patients engulf far less during a 7 day period in vitro than phagocytes from healthy patients

Image from Trouw et al., Mol Immunology (2008) 45:1203

Defective clearance of apoptotic cells

There may be a genetic component to defective apoptosis. Concordance is 25% among monozygotic twins but only 2%

among dizygotic twins suggesting a genetic component HLA-DR2 and HLA-DR3 confer relative risk of 2-5. C1q deficiency results in high likelihood of developing SLE Complement C4a deficiency: 80% of people with SLE have at

least one null allele Can lead to decreased clearance of apoptotic cells and

increased inflammation and presentation of self antigensPatients with SLE may also develop autoantibodies

against adaptor molecules which facilitate phagocytosis of apoptotic cells (C1q, MBL) resulting in defective clearance, classical pathway complement activation, and recruitment of inflammatory cells.

Defective clearance of apoptotic cells

Delayed or defective apoptosis then allows for prolonged exposure of intracellular antigens, “inflammatory cell death phenotype,” inflammatory cell recruitment and presentation of normally protected intracellular components as antigens allowing for autoantibody production

The importance of IFN-

IFN- is able to activate APCs after uptake of self material as well as promote B cell differentiation into plasma cells

IFN- levels appear to correlate with disease severity and levels of anti-DS DNA in SLE

Patients with non-autoimmune diseases treated with IFN- can develop positive ANA, anti-DS DNA abs and occasionally SLE.

Conditions that naturally increase IFN- levels (sunburn, viral infections) can induce SLE flares.

IFN- regulated genes are expressed at higher levels in the blood of SLE patients

Plasmacytoid DCs are the major producers of IFN-. SLE patients have 50-100 fold fewer in circulation as they have migrated to lymph tissues where they remain activated

SNPs in interferon signaling related genes (Tyk2 and interferon regulatory factor 5) also confer increased likelihood of developing lupus

SLE susceptibility polymorphism in STAT4 results in increased sensitivity to IFN- signaling.

The Innate Immune System May Also Play a Role

Toll-Like Receptors recognize molecular patterns (double stranded RNA, DNA, LPS etc) in order to provide rapid response to invading pathogens. They use defined signaling pathways to result in production of inflammatory cytokines and initiate inflammatory reactions.

TLR7 and 9 are selectively expressed on PDCsRegulation in endosomes may regulate

control of NFB vs. IRF7 activation in human plasmacytoid dendritic cells

http://www.sapphirebioscience.com/images/wallcharts/alexis_toll_like_receptors_detail.jpg

Signaling involving IRF7 or IRF 5 (polymorphisms in SLE) can lead to high levels of IFN- production

TLRs are thought to also recognize self antigens in the context of inflammatory

diseases

All TLRs (except 5 and 10) have been shown to be activated by endogenous molecules in the context of cell death

TLR7 and 9 are expressed only in endosomes to decrease the chance of coming in contact with endogenous RNA or DNA

TLR7 and 9 are activated by DNA/anti-DNA IgG complexes resulting in IFN- and autoantibody production.

However, immune complexes are taken up by cells with FcRIIa and taken to the endosome where they can activate TLR 7 and 9. This Results in

signaling cascade activation that increases production of IFN-

Image taken from Barrat and Coffman 2008

B-cell Disregulation

This contributes to the disregulation of the B-cell: increased levels of IFN-differentiate B-cells into antibody-producing plasmocytes and upregulates

B-cell survival factors such as BAFF.

Image taken from Barrat and Coffman 2008

Additionally, recent identification of a genetic linkage of an allele that suppresses B-lymphocyte kinase levels in SLE emphasizes the importance of regulation of B cell proliferation and tolerance

Mechanism Summary

Defects in clearance of apoptotic cells can lead to exposure of intracellular immunogenic components which can be taken up by DC and presented to autoreactive B cells (made this way during random somatic hypermutation).In the right genetic environment, these B cells may become activated to produce autoantibodies.Polymorphisms or mutations in genes in numerous steps of B-cell regulation or IFN-responsiveness can predispose to SLE

(FcRIIa, IRF5, STAT4, BLK)

Mechanism Summary

Once autoantibodies (particularly anti-DS DNA) are present, they can complex with DNA exposed on dying cells and then bind to the FcRIIa on PDCs, activate TLR 7 and 9, and result in high levels of IFN- production.

IFN- encourages a feed-forward mechanism of continued plasma cell activation to produce increased amounts of autoantibodies and encourage further disease progression and tissue destruction.

Autoantibodies

ANA: against targets in the nucleus, but only those which have intrinsic immunological activity: i.e.. They can activate the innate immune system via Toll-like receptors

Anti DS-DNA in particular recognizes DNA in complex with nucleosome components (histone-derived peptides in particular)

Can correlate with nephritis Immune complexes with anti-DNA ab/DNA can increase the expression of IFN-

via plamacytoid dendritic cells Anti-Sm: detects ribonucleoproteins involved in processing of

mRNA; doesn’t track with disease, specific for lupus SSA/Ro and SSB/La: detect ribonucleoproteins, associated with

SICCA syndrome and photosensitivity Anti NMDA to subunits NR2a and NR2b may be associated with

neuropsychiatric symptoms “Antiphospholipid” antibodies are ab against phospholipid-

binding proteins or phospholipids that are prothrombogenic. Ex: lupus anticoagulant, anticardiolipin, and anti beta2-glycoprotein I

Why are autoantibodies so bad?

Renal disease IgA, IgM, IgG and complement deposition in the

mesangium and subendothelial and subepithelial of the GBM that results in complement activation and recruitment of inflammatory cells that result in tissue destruction.

Cross reactivity of anti-DS DNA antibodies with -actinin may also result in a direct focusing of complement activation

Skin disease Inflammation and breakdown of the dermal-epidermal

junction. UV exposure can worsen because it promotes apoptosis in

the skin resulting in autoantibody binding and tissue injury via complement activation or inflammatory cell activation

Anti-Ro antibodies are associated with skin flares

Why are autoantibodies so bad?

In the CNS, vasculitis is rare Anti-NMDA receptor antibodies may contribute to

cerebral lupus phenotypes See more microinfarcts and degeneration or

proliferative changes in blood vessels, thought to be related to IC deposition

Antiphospholipid abs may contribute to thrombotic events anywhere in the body aPLs bind to endothelial cells, monocytes, neutrophils

and platelets causing inflammation and procoagulant release

This process is dependent on complement activation

How Understanding the Mechanism of SLE will influence therapy

Currently, general immunosuppressants and antimalarials are the therapy of choice for lupus (steroids, plaquenil) and lupus nephritis (cyclophosphamide –cellcept may become an approved option)

Current therapies are limited by side effects No new FDA approved drug for lupus have

surfaced in 40 years!Research into the underlying mechanisms will

allow for more directed therapies that may provide better control of SLE with fewer side effects

How Understanding the Mechanism of SLE will influence therapy

Removal of B cells may improve disease control Open label trials of rituximab (anti CD-20) have shown up to 80%

response, 50% with sustained response after 12 months. A recent RCT (EXPLORER) did not show a benefit with rituximab but patients were very sick and both control and study patients received high doses of steroids which may have undercut the benefit seen in patients given rituximab

Trials of anti-IFN- antibodies are underway Preliminary trials of inhibitory DNA sequences to block

immune complex binding to TLRs suggest that preventing aberrant TLR 7 and 9 activation decreases IFN-levels and disease flares

Anti-IL-10 trials are ongoing. Preliminary trials suggest improvement in skin and joint symptoms

Trials are also ongoing to block other B-cell stimulating signals (anti-BlyS=Belimumab, atacicept (soluble fusion protein that inhibits Blys ligand) and to block helper T cell activation.

Summary

Lupus is a disease of autoantibody formation that results in varied clinical manifestations

Disregulation of apoptosis, B-cell survival and proliferation and IFN- production appear to be the major inciting events

Ongoing research into the mechanisms which lead to SLE will hopefully provide us with novel effective therapies with improved side effect profiles

References

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Barrat, F.J. and R. Coffman., “Development of TLR inhibitors for the treatment of autoimmune diseases,” Immunological Reviews (2008) 223:271-283.

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Patients In Vivo,” Journal of Immunology (2009) 182: 34 – 38. Karp, David R. “Complement and Systemic Lupus Erythematosus,” Curr Opin Rheumatol (2005) 17:538-542. Klippel, John H., Primer on the Rheumatic Diseases. Thirteenth Edition., 2008 Springer Science +Business

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Systemic Lupus Erythematosus,” Arthritis Rheum (2007); 57:585-592. Manzi, Susan., “Lupus Update: Perspective and clinical pearls,” Cleveland Clinic Journal of Medicine (2009),

76(2):137-142. Means, T.K. and Luster, A.D., “Toll-like Receptor Activation in the Pathogenesis of Systemic Lupus

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(2002) 2:945 – 956. Rahman, Anisur and David Isenberg., “Systemic Lupus Erythematosus,” N Engl J Med. (2008) 358:9 929-39. Ramsey-Goldman et al., “Frequency of fractures in women with systemic lupus erythematosus: comparison with

United States population data,” Arthritis Rheum (2005) 52: 148-1490 Trouw, L.A. et al., “ Role of complement and complement regulators in the removal of apoptotic cells.” Mol

Immunology (2008) 45:1199-1207. Weening, J.J. et al., “The Classification of Glomerulonephritis in Systemic Lupus Erythematosus Revisited,” J Am

Soc Nephrol (2004) 15: 241–250. Wentworth and Davies., “Systemic Lupus Erythematosus,” Nature Reviews Drug Discovery (2009) 8:103-104. www.emedicine.medscape.com

Kids!!!