SLE – An Overview and Update
Pathobiology Course – JAN 24, 2014
Emilio B. González, MD
Professor and Director, Rheumatology
UTMB
Systemic Lupus Erythematosus (SLE)
• Definition: a chronic inflammatory systemic
autoimmune disease of unknown etiology
characterized by polyclonal B-cell activation and
abnormal autoantibodies
• Not one disease but several clinical subsets, some
mild, e.g., “skin and joint” lupus, and others more
severe, with profound thrombocytopenia,
thrombosis from APS (antiphospholipid syndrome),
and severe renal, lung, and CNS involvement
1982 ACR (Revised 1997) SLE
Classification Criteria
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Malar (butterfly) rash
Discoid lesions
Photosensitivity
Oral ulcers
Non-deforming arthritis (non-erosive for the most part)
Serositis: pleuropericarditis, aseptic peritonitis
Renal: persistent proteinuria › 0.5 g/d or ›3+ or cellular casts
Neurologic disorders: seizures, psychosis
Heme: hemolytic anemia; leukopenia, thrombocytopenia
Immune: anti-DNA, or anti-Sm, or APS (ACA IgG, IgM), or lupus
anticoagulant (standard) or false + RPR
Positive FANA (fluorescent antinuclear antibody)
Definite SLE = 4 or more positive criteria
New SLICC* Revision of the ACR
Classification Criteria - Clinical
1.
2.
3.
4.
5.
Acute/subacute cutaneous lupus
Chronic cutaneous lupus
Oral/Nasal ulcers
Nonscarring alopecia
Inflammatory synovitis with physician-observed swelling of two or more
joints OR tender joints with morning stiffness
6. Serositis
7. Renal: Urine protein/creatinine (or 24 hr urine protein) representing at
least 500 mg of protein/24 hours or red blood cell casts
8. Neurologic: seizures, psychosis, mononeuritis multiplex, myelitis,
peripheral or cranial neuropathy, cerebritis (acute confusional state)
9. Hemolytic anemia
10. Leukopenia (< 4000/mm3 at least once) OR Lymphopenia (<
1000/mm3 at least once)
11. Thrombocytopenia (<100,000/mm3) at least once
*Systemic Lupus International Collaborating Clinics (SLICC)
SLICC Revision of the ACR
Classification Criteria – Immunologic
1.
ANA (antinuclear antibody) above laboratory reference range
2. Anti-dsDNA above laboratory reference range (except
ELISA: twice above laboratory reference range)
3. Anti-Sm (anti-Smith) antibody
4. APS abs: LAC, false-positive test for syphilis, anticardiolipin
IgG, IgM, or IgA Abs, at least twice normal or mediumhigh titer, same for anti-B2 glycoprotein 1
5. Low complement: low C3, low C4, low CH50
6. Direct Coombs test in the absence of hemolytic anemia
Petri M, et al. A & R, 2012
Lupus - SLICC* 17 New Classification
Criteria: 4 needed
• At least 1 clinical plus at least 1 immunologic
criteria (for a total of 4)
or
• Lupus nephritis by biopsy as the sole clinical
criterion plus SLE autoantibodies: (+) ANA or
(+) anti-dsDNA
*Systemic Lupus International Collaborating Clinics (SLICC)
Petri M, et al. A & R, 2012
The Use of ANA for Screening
• Anti-nuclear antibody (ANA) is considered a
screening method for diagnosis of autoimmune
disorders
• Immunofluorescence ANA assay (IF) remains
the gold standard for detection of ANA (2011
ACR position statement)
• Many laboratories perform immunoassays (such
as the multiplexed immunobead assay), for the
detection of ANA as it is less labor-intensive
Our UTMB Data - AIMS
• To compare ANA detection by multiplex
immunobead assay with the gold standard
immunofluorescence (IF) at UTMB
• Patient samples tested for both assays: 110
• Multiplex immunobead assay (MIA) were
considered positive based on the manufacturer’s
instructions
• Immunofluorescence (IF) was considered
positive at a titer ≥ 1:160
Methods
• Data collected prospectively on rheumatology patients from
March 2011 to May 2012 tested for ANA by multiplex
immunobead assay MIA (BioPlex ANA screen, Bio-Rad
Laboratories, Hercules, CA, USA) and IF assay (HEp-2000
(Immuno Concepts, Sacramento, CA, USA)
• Patients were separated into 4 groups based on positive
and negative ANA by MIA and IF assay
• Data collected by individual chart review including age,
gender, ethnicity, and indication for ANA testing
• Performance characteristics of the immuno-assay were
determined using the IF results as the “gold standard”
Baseline Demographic Comparison
Multiplex +, IF+
(TP)
Multiplex-, IF(TN)
Multiplex+, IF(FP)
MultiplexIF+ (FN)
Age (mean±SD)yrs
45±13
48±15
47±18
48±16
Females n (%)
11 (91%)
59 (79%)
8 (89%)
13 (86%)
Ethnicity*(%)
AA (16%)
C (25%)
H (50%)
AA (13%)
C (70%)
H (9%)
AA (33%)
C (44%)
H (11%)
AA (46%)
C (46%)
SLE
5
1
1
Sjogren’s
1
RA
1
9
1
Reason for testing/Clinical
diagnosis (n)
Systemic sclerosis/PM/DM
2
2
Polyarthralgias
2
Undifferentiated connective
tissue disorder
2
Others
2
30
2
6
3
32
5
1
Dang N, Harper BE, Gonzalez EB, Pierangeli SS, Parekh TM, Loeffelholz M, Bufton KK. Real world experience comparing multiplex immunobead assay
vs immunofluorescence assay for anti-nuclear antibody detection at a university hospital. Abstract 1405, ACR Annual meeting, Washington, D.C,
Nov 2012, S605
Comparison of ANA MIA & IF
IF positive (≥1:160)
IF negative
Multiplex positive n (%) 12 (10%) TP
9 (8%) FP
Multiplex negative n (%) 15 (14%) FN
74 (67%) TN
Sensitivity: 44%
Specificity: 89%
Positive predictive value (PPV): 57%
Negative predictive value (NPV): 83%
Conclusions
• Patients tested negative by the MIA (bioplex) included
patients with definite ANA-associated autoimmune diseases
• These data suggest that screening with an immunoassay
would result in misclassification and potential delay or missed
diagnoses of certain systemic autoimmune diseases Multiplex immunobead assay unreliable
• Immunofluorescence (IF) should remain the preferred assay
for ANA testing in patients with suspicion of autoimmune
disorders until platforms with sensitivities comparable to IF or
better are developed. IF the preferred method – Endorsed by
the American College of Rheumatology (ACR)
Dang N, Harper BE, Gonzalez EB, Pierangeli SS, Parekh TM, Loeffelholz M, Bufton KK. Real world experience comparing
multiplex immunobead assay versus immunofluorescence assay for anti-nuclear antibody detection at a university hospital.
Abstract 1405, ACR Annual meeting, Washington, D.C, Nov 2012, S605
The Genetics of SLE
SLE – Genetic Susceptibility
MHC Related
• HLA-DR1, 2, 3, 4
• Alleles of HLA-DRB1, IRF5,
and STAT4
• C2 - C4 deficiency
• TNF- polymorphisms
Not MHC Related
• C1q deficiency (rare but highest risk)
• Chromosome 1 region 1q41-43
(PARP), region 1q23 (FcγRIIA,
FcγRIIIA)
• IL-10, IL-6 and MBL polymorphisms
• Chromosome 8.p23.1: reduced
expression of BLK and increased
expression of C8orf13 (B cell tyrosine
kinase), chromosome 16p11.22:
integrin  genes IGAM-ITGAX
• B cell gene BANK1
• X chromosome-linked gene IRAK1
MHC = Major Histocompatibility Complex
The Genetics of Lupus – A Complex
Disease
Immune complex
processing: C1q, C2-4, CRP,
ITGAM, FcGR2A, etc
TLR/type I, IFN pathway:
STAT 1, IRAK1,
TREX1, etc
TLR = Toll-like receptor
IFN = interferon
Immune signal
transduction: HLA-DR, IRF5,
STAT4, BANK1, PTPN22, BLK,
TNFSF4, etc
The Future: Epigenetic alterations and potential biomarkers identified in SLE
Mechanism
Target
Cell Type
Alteration
Consequence
DNA methylation
ITGAL (CD11a)
CD70 (TNFSF7)
CD154 (CD40L)
Perforin
KIR family
CD4 T cells
CD4 T cells
CD4 T cells
CD4 T cells
CD4 T cells
Hypomethylation
Hypomethylation
Hypomethylation
Hypomethylation
Hypomethylation
Increased CD11a expression
Increased CD70 expression and B-cell
costimulation
Increased B-cell costimulation
Increased perforin expression
Increased KIR expression
RUNX3
CD4 T cells
Hypermethylation
Dysregulation of ITGAL (CD11a)
expression
MMP9
CD4 T cells
Hypomethylation
Cellular basement membrane breakdown
CD9
CD4 T cells
Hypomethylation
T-cell activation
Histone
modification
Histone H4
Monocytes
Increased
acetylation
Increased expression of proinflammatory
cytokines
MicroRNA
miR-146a
PBMCs
Underexpression
Type I IFN overproduction
miR-21
CD4 T cells
Overexpression
Downregulation of DNMT1 (indirect)
and thus decreased DNA methylation
miR-148a
CD4 T cells
Overexpression
Downregulation of DNMT1 (direct) and
decreased DNA methylation
miR-125a
PBMCs
Underexpresssion
Increased KLF expression and thus
RANTES overproduction
miR-126
CD4 T cells
overexpression
Downregulation of DNMT1 and
decreased DNA methylation
Increased Interferon Alpha
(IFNα) in Lupus
The signature cytokine for the
disease?
Pascual V, Banchereau J, Palucka KA. The central role of dendritic cells and
interferon-alpha in SLE. Curr Opin Rheumatol. 2003; 15(5):548–556
Is It Lupus or IFN- Side Effects?
IFN  side effects
Lupus clinical features
 Cytopenias
 Anemia
 Arthralgias/myalgias
 Skin rash
 Alopecia
Basically the same
constellation of
signs/symptoms plus
(+) autoantibodies
 (+) autoantibodies
 Fever, malaise/flu-like
syndrome
 Seizures, pneumonitis,
etc
One and the same?
SLE
How Does Tissue Injury Occur?
SLE – Several Pathogenetic Mechanisms
• Immune complex-mediated damage: glomerulonephritis
• Direct autoantibody-induced damage: thrombocytopenia and
hemolytic anemia
• APS-induced thrombosis and pregnancy morbidity
• BLyS (BAFF)-APRIL (B lymphocyte stimulators) overexpression:  IFN, TNF, IL-1, IL-6, IL-17, etc
• Complement-mediated inflammation: CNS lupus (C3a),
hypoxemia, and also anti-phospholipid mediated fetal loss
• Either failure of or abnormal response to normal apoptosis
Lupus – Complement Levels
Patients who are always hypocomplementemic
regardless of clinical disease activity may have
an underlying complement deficiency!
Mortality in Lupus - Bimodal Peaks
Early:
• Increased disease activity
• Infections due to immunosuppression
Late:
• Deaths the result of permanent damage: treatment side
effects, atherosclerosis with CAD and heart attacks,
strokes, pulmonary, end-stage renal disease (ESRD), etc
Urowitz MB et al. Am J Med 1976
Cervera R et al. Medicine 1999
Survival rates significantly improved in patients diagnosed 1980-1992, vs 1950-79
However, survival is significantly worse than in the general population
Uramoto KM, et al. A & R. 1999;42:46-46-50; Bernatsky S, et al. A & R. 2006;54:2550-2557
Coronary Heart Disease in Lupus: Premature or
Accelerated Atherosclerosis
 The prevalence ranges from 6 to 15%
 The incidence of a MI is 5 times higher in lupus than in the
general population
 The risk of adverse cardiovascular outcomes is  by a factor of
7 to 17 in patients with lupus as compared with the
Framingham cohort
 Young women (between ages 35 and 44) are significantly more
likely (52-fold increased risk) to experience an MI if they have
lupus
 Reasons: multifactorial and not explained just by the traditional
CAD risk factors
Ward MM. Arthritis Rheum 1999; 42(2): 338-46; Manzi S et al. Am J Epidemiol 1997; 145: 408-15; Petri M, et al. Am J Med 1992;
93: 513-9; Sturfelt G, et al. Medicine (Baltimore) 1992; 71: 216-23; Esdaile JM, et al. Arthritis Rheum 2001; 44: 2331-7
Leading Causes of Death in SLE
 Active lupus
 Infection
 Cardiovascular disease
SLE - Mortality
Study Site:
Patient #:
Deaths:
California¹
408
144
Toronto²
665
124
Active lupus:
49 (34%)
20 (16%)
19 (15.5%)
Infection:
32 (22%)
40 (32%)
25 (20.5 %)
CV disease:
23 (16%)
19 (15.4%)
32 (26.2%)
1. Ward MM, et al. A&R 1995; 38: 1492-9
2. Abu-Shakra M, et al. J Rheum 1995; 22: 1259-64
3. Jacobsen S, et al. Scand J Rheumatol 1999; 28: 75-80
Denmark³
513
122
SLE
Therapeutic Approaches
Treatment of Lupus
• Vitamin D (an immunomodulator!)
• Hydroxychloroquine (HCQ) (Plaquenil®)
• Corticosteroids – Minimize to the extent possible
• Immunosuppressive agents (MTX, azathioprine,
mycophenolate mofetil, etc)
• Targeted biologic therapies: belimumab (Benlysta®),
rituximab (Rituxan®)
• Statins? especially for APS (antiphospholipid syndrome)?*
*Erkan D, Willis R, Murthy VL, Basra G, Vega J, Ruiz-Limón P, Carrera AL, Papalardo E, Martínez-Martínez LA, González
EB, Pierangeli SS. A prospective open-label pilot study of fluvastatin on proinflammatory and prothrombotic biomarkers in
antiphospholipid antibody positive patients. Ann Rheum Dis. 2013 Aug 9. doi: 10.1136/annrheumdis-2013-203622. [Epub
ahead of print] PMID: 23933625
Every patient with lupus should be on
vitamin D and hydroxychloroquine (HCQ)!
•
A 20-ng/ml increase in the 25 (OH) D level was associated
with a 21% decrease in the odds of having a high disease
activity score
•
Fifteen (15%) decrease in the odds of having clinically
important proteinuria
•
There was no evidence of additional benefit of 25 (OH) D
beyond a level of 40 ng/ml
Petri M, et al. A & R 2013; 65: 1865–1871
Willis R, Jajoria P, Harper BE, Gonzalez EB, Petri M, Akhter E, Fang H, Pierangeli SS, Abstract
691, ACR Annual meeting, Washington, D.C, Nov 2012, S296.
Hydroxychloroquine (HCQ)
•
It prevents thrombotic events in lupus patients. Ongoing randomized
multi-center trial, APS-ACTION, including UTMB
•
HCQ is an anti-platelet agent, inhibiting aPL-induced GPIIb/IIIa
expression; it does not prolong bleeding time
•
It prevents lupus flare-ups and progression of disease, including
lupus nephritis (LUMINA). It prevents diabetes in patients with RA
receiving it
•
It lowers glycemia and lipids (although modestly)
•
It downregulates inflammation at different levels: prostaglandins,
DNA Abs, T cell activation, inhibits intracellular TLR activation (7 &
9), inhibits IFN-a, IL-1 and IL-6 production, protects the annexin-5
anticoagulant shield from aCL, etc
Willis R, Pierangeli S, Alarcon G, Seif A, Reveille JD, González EB, Dang N, Martìnez Martìnez LA,
Papalardo E, Liu J, McGwin G, Vila LM. Effect of hydroxychloroquine on Pro Inflammatory Cytokines and
Disease Activity in SLE Patients: Data from LUMINA (LXXV), a Multiethnic US Cohort. Lupus 2012 Jul;
21(8):830-5. Epub 2012 Feb 17. PMID: 22343096
+
Effect of HCQ therapy (LUMINA) N = 35 pts
Willis R, Pierangeli S, Alarcon G, Seif A, Reveille JD, González EB, Dang N, Martìnez Martìnez LA, Papalardo E, Liu J,
McGwin G, Vila LM. Lupus 2012 Jul; 21(8):830-5. Epub 2012 Feb 17. PMID: 22343096
Biomarker
Before Rx/median
After Rx/median
p-value
IL6 (pg/mL)
10.68
5.79
0.7956
IL8 (pg/mL)
22.27
16.37
0.9390
VEGF (pg/mL)
164.29
176.97
0.3797
MCP1 (pg/mL)
665.96
738.97
0.5361
IP10 (pg/mL)
525.85
556.81
0.7913
sCD40L (pg/mL)
3053.52
1241.83
0.9027
IFNα(pg/mL)
573.06
381.03
0.2507
IL1 β(pg/mL)
0.00
0.00
0.2645
TNFα(pg/mL)
9.10
7.55
0.8663
aCL IgG (GPL)
9.09
9.60
0.5996
aCL IgM (MPL)
3.04
3.28
0.8870
aCL IgA (APL)
0.12
0.11
0.9096
9
7
0.0157
SLAM-R
Strong positive correlation between the decreases observed in IFN-a and SLAM-R after
HCQ therapy (Spearman correlation coefficient 0.614, p = 0.0087)
Fluvastatin Pilot Trial - 41 Patients,
24 completing the study
NCT00674297
Two centers:
• Hospital for Special Surgery (HSS), New York, NY
• UTMB, Galveston, TX
Four groups:
1.Primary APS (PAPS)
2.SLE /aPL positive
3.Secondary APS (SAPS): SLE + APS
4.Persistently positive aPL
Study Protocol
Baseline
Fluvastatin
40 mg daily
Follow up at 1,
2, 3 months
At 5th month
fluvastatin stopped
Final analysis at 6th
month
Preliminary
Analysis
Effects of Fluvastatin on Levels of Biomarkers in aPL(+) Patients
Biomarkers
(pg/ml)
IL8
IL6
VEGF
IP10
sCD40L
INFα2
IL1β
TNFα
sTF
sICAM-1
sVCAM-1
E-selectin
* P value <0.0001
# of patients with
decreased biomarker
level after fluvastatin
(%)
3/8(38%)
14/17(82%)*
6/17(35%)
8/39(21%)
6/36(17%)
6/13(46%)
11/18(61%)*
10/20(50%)*
22/39(52%)*
18/18(100%)*
11/11(100%)
11/11(100%)
% of maximum
biomarker reduction
with fluvastatin/mean ±
SD
Mean Time (Days) to
maximum biomarker
Level Reduction with
fluvastatin
73.8 ± 31.0
70±34
72.9±32.1
51±24
59.7±23.4
30±23
55.4 ± 23.9
45±22
68.0 ± 21.0
30
81.0 ± 25.6
30
70.3± 30.0
51±28
53.3±30.8
54±28
57.0 ± 29.9
56±25
55.9±35.9
68±25
49.9±34.6
46±32
52.9±26.1
33±19
Summary of Results
Elevated biomarkers in
persistently aPL-positive
patients;
–
–
–
–
–
–
–
–
–
IL6
VEGF
IP10
sCD40L
INFα2
IL1β
TNFα
sTF
sICAM-1
Fluvastatin 40 mg daily for 3
months reduced the levels of
the following biomarkers in
persistently aPL-positive
patients
–
–
–
–
–
–
IL1β
VEGF
TNFα
IP10
sCD40L
sTF
Fluvastatin significantly and reversibly reduced the levels of 6/12 (50%)
biomarkers (IL1β, VEGF, TNFα, IP10, sCD40L and sTF)
Erkan D, Willis R, Murthy VL, Basra G, Vega J, Ruiz-Limón P, Carrera AL, Papalardo E, Martínez-Martínez LA,
González EB, Pierangeli SS. A prospective open-label pilot study of fluvastatin on proinflammatory and
prothrombotic biomarkers in antiphospholipid antibody positive patients. Ann Rheum Dis. 2013 Aug 9. doi:
10.1136/annrheumdis-2013-203622. [Epub ahead of print] PMID: 23933625
NSAIDS and Steroids
New FDA-Approved Agent –
Belimumab (Benlysta®)
• Anti-BLYS humanized monoclonal antibody.
Ongoing Phase IV trials in African-American
patients (multi-center trial including UTMB)
• Problematic indications: not for thrombocytopenia,
CNS, or renal lupus
• Helpful but modest efficacy
• It helps reduce steroids, prevent flares, and
maintain disease remission!
Belimumab Significantly Reduced
Anti-dsDNA By Week 4
15% of anti-dsDNA positive subjects treated with belimumab
converted to negative compared to 3% of placebo patients
p=0.0296 week 24; p=0.0015 week 52; anti-dsDNA+ defined as ≥ 30 IU/mLby ELISA
Updated data through 76 weeks to be presented at ACR on 11/14: Abstract #1985 Stohl et al.
Belimumab LBSL02 phase 2 SLE study
The Future - Biomarkers and Targeted Therapies
• Develop better biomarkers for flares and predictors of
response
• Corticosteroid-free regimens
• Other B cell blockers, e.g., ocrelizumab, epratuzumab,
TACI-Ig (atacicept, an anti-BLyS/April agent). Ongoing trials
• Interferon alpha (IFN) blockers, e.g., sifalimumab. Good
promising data. Ongoing trials
• Anti-C5: humanized monoclonal Ab, especially for APS,
ongoing trials, including UTMB
• Interferon gamma (IFNγ) blockers: for renal lupus. Ongoing
trials
Petri M, et al. Sifalimumab, a human anti-IFN alpha antibody in SLE. A & R 65; 2013: 1011-21
FIN
Questions?