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ARTHRITIS & RHEUMATISMVol. 62, No. 12, December 2010, pp 3730–3740DOI 10.1002/art.27700© 2010, American College of Rheumatology
Anti–U1 RNP Antibodies in Cerebrospinal Fluid AreAssociated With Central Neuropsychiatric Manifestations
in Systemic Lupus Erythematosus andMixed Connective Tissue Disease
Takeshi Sato,1 Takao Fujii,2 Tomoko Yokoyama,2 Yoshimasa Fujita,3 Yoshitaka Imura,4
Naoichiro Yukawa,2 Daisuke Kawabata,2 Takaki Nojima,2 Koichiro Ohmura,2
Takashi Usui,2 and Tsuneyo Mimori2
Objective. To determine the significance ofanti–U1 RNP antibodies in the cerebrospinal fluid(CSF) of patients with systemic lupus erythematosus(SLE) or mixed connective tissue disease (MCTD) whohave central neuropsychiatric SLE (NPSLE).
Methods. The frequency of antinuclear antibodiesincluding anti–U1 RNP antibodies in the sera and CSFof 24 patients with SLE and 4 patients with MCTD, allof whom had neuropsychiatric syndromes, was deter-mined using an RNA immunoprecipitation assay and anenzyme-linked immunosorbent assay. The frequency ofanti–U1 RNP antibodies in the CSF of patients withcentral NPSLE was examined, and the anti–U1 RNPindex ([CSF anti–U1 RNP antibodies/serum anti–U1RNP antibodies]/[CSF IgG/serum IgG]) was comparedwith CSF interleukin-6 (IL-6) levels and the albuminquotient (Qalb, an indicator of blood–brain barrierdamage). CSF and serum antibodies against U1-70K,U1-A, and U1-C, including autoantigenic regions, were
examined, and the U1-70K, U1-A, and U1-C indices aswell as the anti–U1 RNP index were calculated.
Results. CSF anti–U1 RNP antibodies with anincreased anti–U1 RNP index showed 64.3% sensitivityand 92.9% specificity for central NPSLE. The anti–U1RNP index did not correlate with CSF IL-6 levels or theQalb. The anti–U1-70K index was higher than theanti–U1-A and anti–U1-C indices in the CSF of anti–U1RNP antibody–positive patients with central NPSLE.The major autoantigenic region for CSF anti–U1-70Kantibodies appeared to be localized in U1-70K aminoacid 141–164 residue within the RNA-binding domain.
Conclusion. The frequency of anti–U1 RNP anti-bodies in the CSF and the anti–U1 RNP index are usefulindicators of central NPSLE in anti–U1 RNP antibody–positive patients. The predominance of anti–U1-70Kantibodies in CSF suggests intrathecal anti–U1 RNPantibody production.
Systemic lupus erythematosus (SLE) is a chronic,remitting and relapsing, multisystem autoimmune dis-ease that predominantly affects women. Neuropsychiat-ric SLE (NPSLE) involving the central nervous system(CNS; central NPSLE) is a severe, life-threatening man-ifestation. The overall prevalence of NPSLE varied from14% to 75% in a representative selection of studies usingthe American College of Rheumatology (ACR) nomen-clature (1). In fact, NPSLE may affect mortality in SLEand is associated with a significant negative impact on apatient’s quality of life. However, 41% of all CNSmanifestations in patients with SLE may be attributed tofactors other than lupus (2).
Although the pathophysiology of most NPSLEcases is not well determined, a particular subset of
Supported by the Japanese Ministry of Education, Culture,Sports, Science, and Technology (Grant-in-Aid for Scientific Re-search) and the Japanese Ministry of Health, Labor, and Welfare(Grant for Intractable Diseases).
1Takeshi Sato, MD: Kyoto University and National HospitalOrganization Utano Hospital, Kyoto, Japan; 2Takao Fujii, MD, PhD,Tomoko Yokoyama, MD, Naoichiro Yukawa, MD, Daisuke Kawa-bata, MD, PhD, Takaki Nojima, MD, PhD, Koichiro Ohmura, MD,PhD, Takashi Usui, MD, PhD, Tsuneyo Mimori, MD, PhD: KyotoUniversity, Kyoto, Japan; 3Yoshimasa Fujita, MD, PhD: KanazawaMedical University, Ishikawa, Japan; 4Yoshitaka Imura, MD: OsakaRed Cross Hospital, Osaka, Japan.
Address correspondence and reprint requests to Takao Fujii,MD, PhD, Department of Rheumatology and Clinical Immunology,Graduate School of Medicine, Kyoto University, 54 ShogoinKawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: [email protected].
Submitted for publication November 7, 2009; accepted inrevised form August 3, 2010.
3730
autoantibodies is associated with neuronal injury. Asubset of anti–double-stranded DNA (anti-dsDNA) an-tibodies cross-reacts with a sequence within theN-methyl-D-aspartate receptor subunit NR2 (anti-NR2antibodies), causing excitatory synaptic transmission inthe CNS (3). Anti-NR2 antibodies preferentially targetthe hippocampus, suggesting that anti-NR2 antibodiesmay disrupt normal cognitive processes (4). However,serum anti-NR2 antibodies may be associated with de-pressive mood (5) but not cognitive dysfunction (5,6).Arinuma et al (7) reported that anti-NR2 antibodies incerebrospinal fluid (CSF), but not in serum, were in-volved in diffuse central NPSLE. Anti–ribosomal Pantibodies have been reported as being neurotoxic au-toantibodies (8–10). Abnormal behavior can be inducedin healthy mice by administering anti–ribosomal P anti-bodies (10), although serum anti–ribosomal P antibodiesare not specific to human central NPSLE (11). Inassociation with thrombosis, anti-�2 glycoprotein I andprothrombin antibodies are recognized as pathogenicantibodies in central NPSLE such as cerebrovasculardisease (12).
In addition to autoantibodies, several proinflam-matory cytokines and chemokines have been detected inthe serum and CSF of patients with SLE. The mostimportant cytokine in SLE and NPSLE may beinterferon-� (IFN�) (13–15). A recent study showed thepresence of IFN�, IFN�-inducible 10-kd protein (IP-10), interleukin-8 (IL-8), and monocyte chemotacticprotein 1 (MCP-1) in CSF from patients with NPSLE(15). The generation of IFN� in patients with SLE iscaused, at least partially, by autoantibodies that bind toRNP particles released from dead and dying cells.Santer et al clearly suggested that IFN-inducing activityin CSF correlates with serum anti–U1 RNP antibodiesbut not with other known antinuclear antibodies(ANAs) (15). Therefore, anti–U1 RNP antibodies andtheir immune complex in CSF may have pathogenicroles in central NPSLE. Okada et al (16) reported 14patients with aseptic meningitis (8 with SLE, 6 withmixed connective tissue disease [MCTD] or undifferen-tiated connective tissue disease, and 1 with Sjogren’ssyndrome) among 1,560 patients with connective tissuedisease. Serum anti–U1 RNP antibodies were positive in13 of the 14 patients with aseptic meningitis, suggestingthat anti–U1 RNP antibodies may be linked to centralneuropsychiatric manifestations (16). In the presentstudy, we evaluated the clinical significance and immu-nologic characteristics of anti–U1 RNP antibodies inCSF derived from patients with SLE and patients withMCTD who had central neuropsychiatric manifestations.
PATIENTS AND METHODS
Patients. Patients with SLE or MCTD who revealedCNS-related disease manifestations and had been admitted tothe Department of Rheumatology and Clinical Immunology,Kyoto University Hospital, from March 2002 to October 2007were enrolled. SLE was diagnosed according to the ACRcriteria (17,18), and MCTD was diagnosed according to thecriteria proposed by the Ministry of Health and Welfare inJapan (19). CNS manifestations were classified according tothe case definitions for neuropsychiatric syndromes in SLE(20). Evaluation of neuropsychiatric syndromes included neu-ropsychiatric testing and magnetic resonance imaging (MRI)of the brain. Central NPSLE was diagnosed retrospectively,according to the criteria described by Kwiecinski et al (21),with some modifications.
Briefly, central NPSLE was defined as the presenceof at least 2 of the following: 1) recent-onset psychosis,2) transverse myelitis, 3) aseptic meningitis, 4) seizures,5) pathologic changes visualized on brain MRI, and 6) severelyabnormal cognitive dysfunction documented by neuropsychi-atric testing. Oligoclonal IgG bands in the CSF were notincluded, and CNS manifestations caused by other factors(e.g., concurrent non-SLE neuropsychiatric diseases such asinfection, uremia, and hypertension, or adverse reactions ofSLE therapy) were not defined as central NPSLE.
In the present study, small punctate focal lesions inwhite matter, cortical atrophy, periventricular white matterhyperintensity, diffuse white matter changes, discrete graymatter lesions, diffuse gray matter hyperintensities, cerebraledema, and new infarct on brain MRI were considered to bepathologic changes. The present study was conducted in com-pliance with the Declaration of Helsinki and was approved bythe Kyoto University Ethics Committee Review Board (ap-proval no. E97).
Samples. After an acute phase of massive cerebrovas-cular disease was ruled out by brain MRI screening, CSFsamples were obtained from patients within 3 days of the onsetof CNS manifestations. Written informed consent was pro-vided by all of the studied patients. Serum samples werecollected from all patients on the same day, and both serumand CSF samples were stored at �80°C. Routine CSF andserum analyses, including total protein, albumin, and IgGlevels, were conducted. CSF IL-6 (R&D Systems) and IFN�(Bender MedSystems) levels were determined by enzyme-linked immunosorbent assay (ELISA), according to the man-ufacturer’s protocol. The sensitivity of the IFN� assay was �5pg/ml.
Albumin quotient (Qalb) and IgG index . To determineblood–brain barrier damage, the albumin quotient (normal�0.0076) was calculated as the ratio of CSF albumin (mg/dl) toserum albumin (mg/dl) (22). The IgG index was calculated asthe IgG ratio, as follows: (CSF IgG concentration [mg/dl]/serum IgG concentration [mg/dl])/Qalb.
Detection of ANAs in sera and CSF by RNA immuno-precipitation assay. An RNA immunoprecipitation assay usingHeLa cell extracts was performed to evaluate the expression ofanti-RNA–associated antigen antibodies, such as anti–U1RNP, anti-Sm, anti-SSA/Ro (anti-SSA), anti-SSB/La (anti-SSB), and anti–ribosomal P antibodies, in serum and CSF (23).A 10-�l sample of serum or CSF was mixed with 2 mg of
ROLE OF ANTI–U1 RNP ANTIBODIES IN NPSLE 3731
protein A–Sepharose CL-4B (GE Healthcare) in 500 �l ofimmunoprecipitation buffer (10 mM Tris HCl [pH 8.0], 500mM NaCl, 0.1% Nonidet P40 [NP40]) and incubated on arotator for 2 hours at 4°C. The IgG-coated Sepharose waswashed 4 times in 500 �l of immunoprecipitation buffer using10-second spins in a microfuge and resuspended in 400 �l ofNET-2 buffer (50 mM Tris HCl [pH 7.5], 150 mM NaCl, 0.05%NP40). For RNA analysis, this suspension was incubated with300 �l of HeLa cell extracts, derived from 6 � 106 cells, on arotator for 2 hours at 4°C. The antigen-bound Sepharose wasthen collected by 10-second centrifugation in a microfuge,washed 4 times with 500 �l of NET-2 buffer, and resuspendedin 270 ml of NET-2 buffer.
To extract bound RNA, 30 �l of 3.0M sodium acetate,15 �l of 20% sodium dodecyl sulfate, and 300 �l of phenol–chloroform–isoamyl alcohol (50:50:1; containing 0.2 gm8-hydroxyquinoline and 40 ml of 0.1M Tris HCl [pH 7.5]) wereadded to the Sepharose beads. After agitation in a vortex mixerand spinning for 1 minute, RNAs were recovered in theaqueous phase after ethanol precipitation and dissolved in 20�l of electrophoresis sample buffer composed of 10M urea,0.025% bromphenol blue, and 0.02% xylene cyanol FF (Bio-Rad) in Tris–borate–EDTA buffer (90 mM Tris HCl [pH 8.6],90 mM boric acid, and 1 mM EDTA). The RNA samples weredenatured at 65°C for 5 minutes and then resolved by 7Murea–10% polyacrylamide gel electrophoresis with silver stain-ing (Bio-Rad). Anti–U1 RNP, anti-Sm, anti-SSA, and anti-SSBantibodies were determined to be positive when U1 RNA,U1–U6 RNA, SSA RNA (Y1–Y5 RNA), and SSB RNA (5Sribosomal RNA, 7S RNA, and Y1–Y5 RNA), respectively,were precipitated.
Detection of ANAs in sera and CSF by ELISA. Serumanti–U1 RNP, dsDNA, SSA, and ribosomal P antibodieswere also determined by ELISA using recombinant U1 RNP(Mesacup RNP-II test; MBL), purified dsDNA (MesacupDNA-II test “ds”; MBL), recombinant SSA (Mesacup-2 testSS-A; MBL), and recombinant ribosomal P proteins (Ribo-somal P ELISA kit; MBL), according to the manufacturer’sprotocol. Anti–ribosomal P and anti-dsDNA antibodies in CSFwere determined to be positive when the experimental titerwas more than that of the mean � 2SD of 10 negative controls.CSF samples obtained from patients with other autoimmunediseases (4 with polyarteritis nodosa, 4 with multiple sclerosis,and 2 with rheumatoid arthritis) were used as negative CSFcontrols for the anti–ribosomal P and anti-dsDNA antibodies.These patients had CNS manifestation and/or CSF abnormal-ities but not NPSLE, and negative results for their serum ANAanalyses were confirmed.
Sera were diluted 1:100, and CSF samples were diluted1:5 using phosphate buffered saline (PBS). To determine thelevels of anti–U1-70K, anti–U1-A, and anti–U1-C antibodies,recombinant U1-70K, U1-A, and U1-C (MBL) along withcoating buffer (pH 9.4; 1 �g/ml) were bound to ELISA plates,and the wells were blocked using 5% bovine serum albumin(BSA) in PBS. Patient sera and CSF containing anti–U1 RNPantibodies were added and incubated at room temperature for2 hours, followed by detection of bound IgG with alkalinephosphatase–conjugated anti-human IgG (Southern Biotech-nology) at an optical density of 405 nm (OD405 nm) in amicrotiter ELISA reader. All assays were performed in tripli-
cate. We confirmed that the OD405 nm values of the CSFexperimental wells were included within the linear range of thepositive control. If a 1:5 dilution was not appropriate, thedilution was changed in such samples, and the obtained titerwas adjusted.
Anti–U1 RNP ratio and anti–U1 RNP index. Arbitraryunits of the anti–U1 RNP antibodies in each sample weredetermined using serum and CSF anti–U1 RNP antibody–positive (defined as 100 units) and anti–U1 RNP antibody–negative standard samples. More precisely, the arbitrary unitsfor serum or CSF anti–U1 RNP antibodies were calculatedusing the following formula: (OD405 nm of experimental well �OD405 nm of anti–U1 RNP antibody–negative standard well) �100/(OD405 nm of anti–U1 RNP antibody–positive standardwell � OD405 nm of anti–U1 RNP antibody–negative standardwell). After obtaining the arbitrary units of the anti–U1 RNPantibody, the anti–U1 RNP ratio and anti–U1 RNP index werecalculated as follows: CSF arbitrary anti–U1 RNP antibodyunits � 5/serum arbitrary anti–U1 RNP antibody units � 100and anti–U1 RNP ratio/IgG ratio, respectively. The differencein the dilutions between serum (�100) and CSF (�5) wasadjusted, and the anti–U1-70K, anti–U1-A, and anti–U1-Cindices were obtained using the same calculation method.
Determination of autoantigenic regions recognized byserum and CSF anti–U1-70K antibodies. To compare theautoantigenic regions in the U1-70K proteins recognized byCSF and serum anti–U1 RNP antibodies, we prepared 22overlapping synthetic peptides (17–24 amino acids) identical tothe U1-70K partial sequences (24) (Figure 4). Synthetic pep-tides (100 �g/ml) with coating buffer (pH 9.4) were bound toELISA plates, and the wells were blocked with 5% BSA inPBS. Serum and CSF specimens obtained from 8 patients withcentral NPSLE were diluted to 100 �g/ml with PBS and thenincubated for 2 hours at room temperature, followed bydetection of bound IgG with alkaline phosphatase–conjugatedanti-human IgG (Southern Biotechnology) at OD405 nm in amicrotiter ELISA reader. All assays were performed in tripli-cate. Positivity was determined as being a value greater thanthe mean � 2SD of the negative control samples obtained frompatients without serum and CSF anti–U1 RNP antibodies.
Statistical analysis. Differences in the frequencies ofANAs in CSF and serum were evaluated by the chi-square testand Fisher’s exact test, as appropriate. Student’s t-test was usedto compare the difference between 2 group means. Pearson’sproduct-moment correlation coefficients were calculated toevaluate the correlation between the anti–U1 RNP antibodyindex and other indicators. P values less than 0.05 wereconsidered significant.
RESULTS
Patient characteristics. Neuropsychiatric syn-dromes were detected in 24 patients with SLE and 4patients with MCTD. All patients were female, and theiraverage age at the onset of CNS manifestations was 34.1years (range 19–58 years). According to the ACR no-menclature (20), the symptoms of central NPSLE exhib-ited by our patients were as follows: headache, cerebro-
3732 SATO ET AL
Tab
le1.
Cha
ract
eris
tics
ofth
epa
tient
sw
ithce
ntra
lneu
rops
ychi
atri
cSL
E*
Patie
nt/a
ge/
diag
nosi
sC
NS
man
ifest
atio
ns
No.
ofce
llsin
CSF
,pe
r�
l
Tot
alpr
otei
nin
CSF
,m
g/dl
IL-6
inC
SF,
pg/m
lIg
Gin
dex
Qal
b,�
103
AN
As
inse
rum
AN
As
inC
SFA
nti–
U1
RN
Pin
dex
Tre
atm
ent
for
CN
Sm
anife
stat
ions
1/32
/SL
EH
014
1.0
0.89
1.9
U1,
DN
A,S
mU
113
.6H
igh-
dose
CS
2/30
/SL
EA
sM,C
gD5
313.
20.
763.
7U
1,D
NA
,SS
A,S
m,
tRN
A,r
iboP
U1,
SSA
12.4
MP
puls
e,hi
gh-d
ose
CS,
CSA
,IV
CY
C
3/29
/MC
TD
AsM
1181
3,24
00.
7215
.7U
1,SS
A,r
iboP
U1,
SSA
9.4
Hig
h-do
seC
S4/
43/M
CT
DPs
y3
491.
50.
777.
2U
1,U
2U
18.
8M
Ppu
lse,
high
-dos
eC
S,IV
CY
C5/
20/S
LE
CgD
,Psy
048
2.3
0.62
8.1
U1,
SSA
,Sm
,ri
boP
U1
7.6
MP
puls
e,IV
CY
C,
DF
PP,r
ituxi
mab
6/40
/MC
TD
AsM
,CgD
4015
583
70.
5930
.3U
1,D
NA
U1
3.0
Hig
h-do
seC
S,IV
CY
C7/
22/S
LE
AC
S,A
sM,C
VD
3213
27.
80.
7720
.4U
1,Sm
,rib
oPU
12.
2H
igh-
dose
CS,
IVC
YC
8/32
/SL
EA
sM14
128
1,19
00.
8018
.6U
1,D
NA
,SS
A,S
SBU
1,SS
A2.
1M
Ppu
lse,
high
-dos
eC
S
9/39
/SL
EM
ovD
,H4
201.
90.
612.
8U
1,SS
AU
12.
1M
Ppu
lse,
high
-dos
eC
S10
/26/
SLE
H,P
sy3
2964
40.
838.
6U
1,SS
AN
egat
ive
ND
Hig
h-do
seC
S11
/58/
SLE
Se,C
gD0
302.
60.
555.
7U
1,SS
AN
egat
ive
ND
Low
-dos
eC
S12
/41/
SLE
Dem
S3
119
ND
0.68
18.6
DN
A,S
SAD
NA
†N
DH
igh-
dose
CS,
IVC
YC
13/5
6/SL
EH
,Se
135
2.0
0.68
4.5
DN
AN
egat
ive
ND
MP
puls
e14
/29/
SLE
AC
S,Ps
y1
52N
D0.
659.
5N
egat
ive
Neg
ativ
eN
DH
igh-
dose
CS,
ritu
xim
ab
*T
heag
eof
the
patie
nt(y
ears
)re
fers
toth
eag
eat
the
onse
tof
the
first
cent
raln
ervo
ussy
stem
(CN
S)m
anife
stat
ions
.The
mea
n�
SDva
lues
for
cell
num
ber
ince
rebr
ospi
nal
fluid
(CSF
),to
talp
rote
inin
CSF
,int
erle
ukin
-6(I
L-6
)in
CSF
,the
IgG
inde
x,th
eal
bum
inqu
otie
nt(Q
alb)
,and
the
anti–
U1
RN
Pin
dex
wer
eas
follo
ws:
8�
13/ �
l,66
�48
mg/
dl,
494
�95
8pg
/ml,
0.71
�0.
10,1
1.1
�8.
3�
103 ,a
nd6.
8�
4.6,
resp
ectiv
ely.
AN
As�
antin
ucle
aran
tibod
ies;
H�
intr
acta
ble
head
ache
asso
ciat
edw
ithsy
stem
iclu
puse
ryth
emat
osus
(SL
E);
CS
�co
rtic
oste
roid
s;A
sM�
asep
ticm
enin
gitis
;C
gD�
cogn
itive
dysf
unct
ion;
tRN
A�
tran
sfer
RN
A;
ribo
P�
ribo
som
alP;
MP
�m
ethy
lpre
dnis
olon
e;C
SA�
cycl
ospo
rin
A;I
VC
YC
�in
trav
enou
scy
clop
hosp
ham
ide;
MC
TD
�m
ixed
conn
ectiv
etis
sue
dise
ase;
Psy
�ps
ycho
sis;
DF
PP�
doub
le-f
iltra
tion
plas
map
here
sis;
AC
S�
acut
eco
nfus
iona
lsta
te;C
VD
�ce
rebr
ovas
cula
rdi
seas
e;M
ovD
�m
ovem
ent
diso
rder
;ND
�no
tde
term
ined
;Se
�se
izur
esan
dse
izur
edi
sord
ers;
Dem
S�
dem
yelin
atin
gsy
ndro
me.
†A
nti–
doub
le-s
tran
ded
DN
Aan
tibod
ies
inC
SFw
ere
dete
rmin
edby
enzy
me-
linke
dim
mun
osor
bent
assa
yus
ing
nega
tive
cont
rols
(see
Patie
nts
and
Met
hods
).
ROLE OF ANTI–U1 RNP ANTIBODIES IN NPSLE 3733
vascular disease, cognitive dysfunction, seizures andseizure disorder, aseptic meningitis, psychosis, acuteconfusional state, demyelinating syndrome, anxiety dis-order, and movement disorder.
CSF findings and ANA profiles in patients withcentral NPSLE. Central NPSLE was diagnosed in 14patients (Table 1). All patients except patient 11 neededhigh-dose corticosteroid treatment along with methyl-prednisolone pulse therapy and/or immunosuppressiveagents for the treatment of central NPSLE. Aseptic menin-gitis occurred most frequently (5 patients). Among the14 patients with central NPSLE, the cell number in CSFwas increased in 6 patients (46%; normal �4 cells/�l),and the total protein concentration was increased in 8patients (57%; normal �40 mg/dl). The level of IL-6 inCSF was elevated in 5 (42%) of 12 patients (normal�4.3 pg/ml [25]), and the IgG index was increased in 9(64%) of 14 patients (normal �0.67). The Qalb, an indexof blood–brain barrier permeability, was increased in 8(57%) of 14 patients (normal �0.0076 [22]).
Figure 1. Antinuclear antibody (ANA) detection in serum (S) andcerebrospinal fluid (CSF; C) by RNA immunoprecipitation assay.Anti–U1 RNP and anti-SSA/Ro antibodies precipitated U1 RNA andSSA RNA, respectively. A, ANAs in serum and CSF samples fromrepresentative patients with central neuropsychiatric systemic lupuserythematosus (NPSLE). B, ANAs in serum and CSF samples fromrepresentative patients without central NPSLE. Pt � patient; T � totalRNA; tRNA � transfer RNA.
Figure 2. A, ANA frequency in CSF from patients with and those without centralNPSLE. The frequency (%) of CSF ANA positivity in serum ANA–positive patientsis shown. Anti–U1 RNP and anti-SSA antibodies (Ab) were determined by RNAimmunoprecipitation assay, and anti–double-stranded DNA antibodies were deter-mined by enzyme-linked immunosorbent assay. Anti-SSB/La and anti–ribosomal Pantibodies were not detected in CSF. B, Comparison of the IgG and anti–U1 RNPratios. C and D, Correlations between the anti–U1 RNP index and the interleukin-6(IL-6) level or the albumin quotient (Qalb) (C) and between the anti–U1 RNP ratioand the IgG index (D). Pearson’s product-moment correlation coefficients (r) werecalculated in 10 patients with CSF anti–U1 RNP antibody positivity. See Patients andMethods for the formulas to determine the IgG ratio, the anti–U1 RNP ratio, theQalb, the IgG index, and the anti–U1 RNP index. NS � not significant (see Figure1 for other definitions).
3734 SATO ET AL
Tab
le2.
Cha
ract
eris
tics
ofth
epa
tient
sw
ithou
tce
ntra
lneu
rops
ychi
atri
cSL
E*
Patie
nt/a
ge/
diag
nosi
sC
NS
man
ifest
atio
ns
No.
ofce
llsin
CSF
,pe
r�
l
Tot
alpr
otei
nin
CSF
,m
g/dl
IL-6
inC
SF,
pg/m
lIg
Gin
dex
Qal
b,�
103
AN
As
inse
rum
AN
As
inC
SF
Eff
ectiv
etr
eatm
ent
for
CN
Sm
anife
stat
ions
15/2
9/SL
EA
xD1
311.
91.
254.
3U
1R
NP,
DN
A,S
SA†
U1
RN
P,SS
A†
Flu
nitr
azep
am,
clox
azol
am16
/19/
SLE
H1
5520
.11.
387.
4U
1R
NP,
DN
A,S
SAN
egat
ive
NSA
IDs
17/2
4/SL
ESe
020
1.0
0.59
3.1
U1
RN
P,D
NA
,SSA
,Sm
Neg
ativ
eSo
dium
valp
roat
e
18/2
8/SL
EC
gD1
221.
20.
433.
6U
1R
NP,
SmN
egat
ive
Dia
zepa
m19
/49/
SLE
CV
D1
31N
D0.
435.
6U
1R
NP,
DN
A,S
mN
egat
ive
Low
-dos
eas
piri
n
20/2
2/SL
EC
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ROLE OF ANTI–U1 RNP ANTIBODIES IN NPSLE 3735
By RNA immunoprecipitation assay, anti–U1RNP, anti-SSA, anti-SSB, and anti-Sm antibodies weredetected in sera from 11 (79%), 8 (57%), 1 (7%), and 4(29%) of the patients, respectively. Anti–ribosomal Pand anti-dsDNA antibodies were determined to bepositive by ELISA in sera from 4 (29%) and 6 (43%) ofthe patients, respectively. In contrast, anti–U1 RNPantibodies in CSF were most frequently detected byRNA immunoprecipitation assay (82%) in CSF fromanti–U1 RNP antibody–positive patients with centralNPSLE (Figures 1 and 2A). The anti–U1 RNP ratiosincreased more than the IgG ratios in patients with CSFanti–U1 RNP antibodies (Figure 2B). CSF anti-SSA andanti-dsDNA antibodies were detected in only 3 patientsand 1 patient, respectively. Anti–ribosomal P and anti-Sm antibodies were absent in CSF.
CSF findings and ANA profiles in patients with-out central NPSLE. In 14 patients, CNS manifestationswere diagnosed as being concurrent non-SLE centralneuropsychiatric diseases or secondary central neuro-psychiatric syndromes (Table 2). Increased cell numbersand protein concentrations were observed in 3 patients(21%) and 5 patients (36%), respectively. IL-6 expres-sion, the IgG index, and the Qalb were elevated in 4(40%), 7 (54%), and 2 (15%) of the 14 patients,respectively. These values were not significantly differ-ent between patients with and those without centralNPSLE. In contrast to what was observed in patients
with central NPSLE, CSF anti–U1 RNP antibodies weredetected in only 1 patient (patient 15) without centralNPSLE, whereas serum anti–U1 RNP antibodies weredetected frequently (71% of patients).
Correlation of the anti–U1 RNP index with theCSF IL-6 level and the Qalb. The anti–U1 RNP indexwas compared with CSF IL-6 and Qalb values deter-mined from the same CSF samples, obtained from 10anti–U1 RNP antibody–positive patients (patients 1–9and 15). The anti–U1 RNP index was not correlated withIL-6 expression in the CSF (Figure 2C), and the anti–U1RNP ratio was not correlated with the IgG index (Figure2D). The Qalb, which is reportedly a useful blood–brainbarrier permeability indicator, tended to inversely cor-relate with the anti–U1 RNP index (Figure 2C) (y ��0.246x � 9.37).
Dominance of anti–U1-70K antibodies in CSF.Anti–U1 RNP antibodies usually recognize either U1-70K, U1-A, or U1-C proteins, which are the uniquecomponents of the U1 RNP particle. We examined theanti–U1-70K, anti–U1-A, and anti–U1-C indices as wellas the anti–U1 RNP index in 8 CSF anti–U1 RNPantibody–positive patients with central NPSLE (see Ta-ble 1), and the anti–U1 RNP index was �2.0 in allpatients for whom the values were determined. In thesame samples, the anti–U1-70K index was elevated(�1.0) in 7 of 8 patients, whereas the anti–U1-A andanti–U1-C indices were elevated in only a few patients.The average anti–U1-70K index was significantly higherthan the anti–U1-C index and tended to be higher thanthe anti–U1-A index (Figure 3).
U1-70K autoantigenic regions recognized by se-rum and CSF anti–U1 RNP antibodies. More detailedreactivity of the U1-70K protein was examined in seraand CSF from 8 patients with CSF anti–U1 RNPantibodies. All prepared synthetic peptides were recog-nized by the 8 anti–U1 RNP antibody–positive sera,whereas there were 4–21 reacted residues for eachserum sample (data not shown). Serum anti–U1 RNPantibodies in patients with central NPSLE bound theamino acid 61–84 residue most frequently (88%) (Figure4). In contrast, the synthetic peptides that bound CSFanti–U1 RNP antibodies were limited to 20 residues,and there were 1–12 reacted residues for each CSFsample (data not shown). Only amino acid 141–164 residuewas recognized by the majority of the CSF anti–U1 RNPantibodies (88%), whereas the autoepitope patternsrecognized by serum and CSF anti–U1 RNP antibodieswere not significantly different (Figure 4).
Figure 3. Anti–U1 RNP index in 8 patients with central neuropsychi-atric systemic lupus erythematosus (left) and the anti–U1-70K, anti–U1-A, and anti–U1-C indices in the same patients (right). Serum andcerebrospinal fluid anti–U1-70K, anti–U1-A, and anti–U1-C antibodytiters were determined by enzyme-linked immunosorbent assay, usingrecombinant proteins. The patient (Pt) numbers shown correspond tothe patient numbers in Table 1. Because an insufficient quantity ofsample was obtained from patient 1, that index was excluded from thisexperiment. See Patients and Methods for the formula to determinethe anti–U1 RNP, anti–U1-70K, anti–U1-A, and anti–U1-C indices.NS � not significant.
3736 SATO ET AL
DISCUSSION
The results of the present study showed that CSFanti–U1 RNP antibodies determined by RNA immuno-precipitation assay and/or an elevation in the anti–U1RNP index are more specific markers for central NPSLEthan the CSF IL-6 and IgG indices in serum anti–U1RNP antibody–positive patients with SLE or MCTD.This study is the first to show the clinical significance ofCSF anti–U1 RNP antibodies. Although other auto-antibodies such as anti–ribosomal P, anti-NR2, or anti-dsDNA antibodies in association with NPSLE have beendescribed, no serum or CSF anti–U1 RNP antibodieswere reported in either an international cohort or a largestudy (25,26).
Recently, the sensitivity and specificity of a CSFIL-6 level of �4.3 pg/ml for diagnosing central NPSLEhave been reported as 87.5% and 92.3%, respectively(27). However, CSF IL-6 is not specific for disease-
associated NPSLE, because an elevated IL-6 level canalso be caused by infectious meningoencephalitis andcerebrovascular disease. The IgG index is also elevatedin patients without CNS involvement (28), and nostatistically significant differences in the IL-6 level andthe IgG index were observed between central NPSLEand other CNS manifestations in the present study.
An elevated Qalb is strong evidence for blood–brain barrier damage (22), and more than half of ourpatients with central NPSLE had increased blood–brainbarrier permeability, similar to that reported in a previ-ous study (29). However, blood–brain barrier damage iscaused not only by central NPSLE but also by otherfactors. The Qalb was elevated in a patient with drug-induced aseptic meningitis (patient 21) and a patientwith miliary tuberculosis (patient 22).
The presence of anti–U1 RNP antibodies in CSFalong with an anti–U1 RNP index of �2.0 were fre-quently observed in patients with, but not those without,central NPSLE (sensitivity 64.3% and specificity 92.9%).Moreover, the sensitivity and specificity of CSF anti–U1RNP antibodies for central NPSLE in the sera ofanti–U1 RNP antibody–positive patients were calculatedas 81.8% and 90.0%, respectively. Although global pen-etration of serum antibodies into CSF occurs from aserious blood–brain barrier injury, elevation of theanti–U1 RNP index is not influenced, because the IgGratio increases simultaneously in this condition. Thus,the increased anti–U1 RNP antibody titer is a possiblediagnostic marker for CNS manifestations attributableto SLE or MCTD independent of the CSF IL-6 level,IgG index, or Qalb.
In a previous study, a correlation between thepresence of serum anti-Sm antibodies and centralNPSLE was observed (30), whereas the current studydemonstrated no association between the presence ofserum anti–U1 RNP/Sm antibodies and central NPSLE.To date, only our group (31) and a German group ofinvestigators (32) have published case reports of CSFanti–U1 RNP antibody–positive patients. The importantpoint of our study may be the use of an RNA immuno-precipitation assay for detecting anti–U1 RNP anti-bodies. The RNA immunoprecipitation assay is the mostsensitive and specific method among the immunologicmethods used for detecting antibodies, especially thoseagainst RNA or RNA-binding proteins (33). The ab-sence of anti-Sm antibodies may not be attributable to asensitivity problem with the RNA immunoprecipitationassay, because we could not detect the antibodies in thesame samples by ELISA. Because of the lower amountof cellular SSA RNAs than U1 RNAs, SSA RNAs are
Figure 4. Autoantigenic peptide residues recognized by serum andcerebrospinal fluid (CSF) anti–U1-70K antibodies from patients withneuropsychiatric systemic lupus erythematosus (NPSLE). The majorautoantigenic domains for serum and CSF anti–U1-70K antibodieswere examined using enzyme-linked immunosorbent assay with 22overlapping peptides identical to the partial sequence of U1-70K(17–24 amino acids [aa]). Serum and CSF samples obtained from 8patients with central NPSLE were diluted to 100 �g/ml, and positivitywas determined as a value greater than the mean � 2SD of thenegative control samples obtained from patients without serum andCSF anti–U1 RNP antibodies. The data represent the frequency ofpositivity for antipeptide antibodies in 8 serum and CSF samples fromCSF anti–U1 RNP antibody–positive patients with central NPSLE.
ROLE OF ANTI–U1 RNP ANTIBODIES IN NPSLE 3737
more difficult to visualize than U1 RNAs by RNAimmunoprecipitation assay. It is possible that anti–U1RNP antibodies were unequivocally detected in thissystem, and the present study may not indicate thatintrathecal ANA stimulation is specific to anti–U1 RNPantibodies.
The striking deficit in CNS pathology, specificallythe lack of vasculitis or massive cellular infiltrates inpatients dying of central NPSLE, suggests that thepathogenesis differs from that of immune complex dep-osition, which is a characteristic of lupus nephritis.Rather, anti–U1 RNP antibodies may act as an inducerof proinflammatory cytokines. It is worth noting thatsera and CSF from patients with NPSLE show abnor-mally high IFN�-inducing activity (15). In addition toIFN� and immune complex formed by CSF, autoanti-bodies produce significantly increased levels of IP-10/CXCL, IL-8, and MCP-1 (34–36), and this phenomenonis most distinguished in the sera of anti–U1 RNPantibody–positive patients (15). It is interesting that thishypothesis was demonstrated, because IFN� is stronglyinduced by U1 RNP–containing immune complex (37)and is the key cytokine involved in the pathogenesis ofSLE. Immune complex and IFN� were not detected inmost of the CSF samples from our patients (data notshown), possibly because CSF IFN� was degradatedquickly, and immune complex moved to Fc�-expressingcells.
Thus far, it has been reported that a certain ANAsubset is relevant to central NPSLE. First, serum anti–ribosomal P antibodies are definitely a useful diagnosticmarker for SLE (38); however, it is controversialwhether anti–ribosomal P antibodies in serum and/orCSF are a link to central NPSLE (11,38), whereas thepresence of ribosomal P protein on the endothelial cellshas been demonstrated (39). It is likely that anti–ribosomal P antibodies might not be able to pass effec-tively through the blood–brain barrier due to binding toCNS endothelial cells (8). When paired serum and CSFsamples were diluted to the same IgG concentrationsand used for Western blotting, selective enrichment ofIgG anti–ribosomal P occurred in the CSF of a fewpatients (39). Anti–ribosomal P antibodies were notdetected in CSF from our patients.
Kowal et al reported that a certain anti-dsDNAantibody subset in CSF which cross-reacts with the NR2glutamate receptor causes apoptotic neuronal death inthe mouse hippocampus (3). Although the CSF level ofanti-NR2 antibodies was higher in patients with centralNPSLE than in other SLE groups, the highest CSFanti-NR2 antibody levels have been detected in patients
with septic meningitis (26), suggesting that anti-NR2antibodies attack neurotransmitters directly through abreach in blood–brain barrier integrity. Although serumand CSF anti-NR2 antibodies were not investigated inthe present study, the anti-NR2 antibody–mediated neu-ronal diseases are different from aseptic meningitis andappear to be associated with anti–U1 RNP antibodies.Unfortunately, the present study did not clearly indicatethe types of central NPSLE with which anti–U1 RNPantibodies are most associated. In accordance with aprevious study (27), our data show no significant corre-lation between the presence of serum or CSF anti-SSAantibodies and central NPSLE.
The presence of ANAs in CSF can be explainedby the following 3 mechanisms: 1) in situ antibodyproduction in the CNS, 2) a blood–brain barrier breachthat would allow antibodies to cross a normally re-stricted compartment, and 3) an increased antibodyconcentration resulting from a reduced CSF flow rate.However, our data strongly suggest intrathecal produc-tion of anti–U1 RNP antibodies, because the anti–U1-70K, anti–U1-A, and anti–U1-C indices were not equallyelevated, and the anti–U1 RNP and anti–U1-70K indicesin most patients increased by �2.0. Even if a blood–brain barrier breach or reduced flow rate of CSF oc-curred, it is unlikely that either anti–U1-70K, anti–U1-A, or anti–U1-C antibodies penetrated or movedoutside the CSF. The observation that a different U1-70K peptide recognition pattern by serum and CSFanti–U1-70K antibodies is evident also suggests intrathe-cal anti–U1 RNP antibody production.
Notably, the autoantigenic amino acid 141–164residue for CSF anti–U1-70K antibodies in patients withcentral NPSLE was located within the RNA-bindingdomain (amino acids 92–202), including T cell (40) andB cell (41–44) major epitopes of human anti–U1-70Kantibodies. Guldner et al identified the amino acid56–195 domain as the major antigenic epitope recog-nized by all tested sera (41). Cram et al reported theamino acid 100–156 residue as one of the major epitopesin the human 70K protein (42). James et al showed thatthe basic amino acid–rich sequences are the early auto-antigenic determinants of the 70K C-terminus (45).More detailed experiments using a large number of CSFsamples are necessary to clarify the immunologic char-acteristics of intrathecal anti–U1 RNP antibodies,whereas our results suggest the possibility that ANAproduction in CSF is stimulated by an antigen-drivenmechanism.
In conclusion, CSF anti–U1 RNP antibodies,which may be produced in the CNS, are a clinically
3738 SATO ET AL
useful indicator of central NPSLE. However, the presentstudy has some limitations. First, the usefulness of theanti–U1 RNP index is limited to patients with NPSLEwho have serum anti–U1 RNP antibodies. Second, theRNA immunoprecipitation method may be required todetermine a low anti–U1 RNP antibody titer. Finally,our results were observed in a small number of Japanesepatients. A more detailed association of CSF anti–U1RNP antibodies with other humoral factors or activatedneuronal cells in the CNS should be elucidated in afuture study.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Fujii had full access to all of thedata in the study and takes responsibility for the integrity of the dataand the accuracy of the data analysis.Study conception and design. Fujii, Yokoyama, Yukawa, Nojima,Mimori.Acquisition of data. Sato, Fujii, Yokoyama, Fujita, Imura, Usui.Analysis and interpretation of data. Sato, Fujii, Kawabata, Ohmura.
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