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Nucleolar antigen specific for antinucleolar antibody in the sera of patients with systemic rheumatic disease.

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Nucleolar Antigen Specific for Antinucleolar Antibody
In the Sera of Patients With Systemic Rheumatic Disease
Shoji Miyawaki and Robert F. Ritchie
Phenol extractable nucleolar 7s RNA produced precipitin reactions in gel
with sera containing high titers of antinucleolar antibodies (ANoA) and
strongly adsorbed serum ANoA activity. Similar extracts from nuclei or
cytoplasm lacked this ability. Extracted and single-stranded DNA also
precipitated with ANoA but was only slightly able to absorb serum ANoA
activity. Precipitating ANoA was usually found in patients with systemic
sclerosis or Raynaud's phenomenon but was also found in one-third of
patients with other rheumatic diseases.
Recent studies have identified a host of antibodies to nuclear constituents in the sera of
patients with systemic lupus erythematosus
(SLE) and other systemic rheumatic diseases.
These include antibodies to native doublestranded DNA (DS-DN.4) (1.2). denatured
single-stranded DS.\(SS-DNA)
( 3 , 4 ) . nucleohistone ( 5 ) , histone (6), acidic nuclear protein (7), cytoplasmic ribosomes (8,9), doublestranded RNA. and polynucleotides (10. 11). In
addition, antibodies to ribonucleoprotein have
been identified more recently (12, 13).
Several studies have been directed to the occurrence of an antibody to the nucleolus
(ANoA) in some patients with systemic rheumatic diseases since the first description by Beck
in 1961 (14). Because of technical problems.
identification of the nucleolar antigen has been
difficult. T h i s paper reports the identification of
nucleolar antigen and methods of isolation. Recently. similar findings on antigen extracted by
different techniques has been presented (1 5).
MATERIALS AND METHODS
Serum Specimens. Sixtecn sera were selevtcd k a u s e
of high .-\No.%
titers. Thirty-six additional sera were se-
l m e d from patients with SLE. I2 from patients with the
procainamide-induced lupus (pronesty1 lupus) and 17 fmm
patients with rheumatoid arthritis (Rh). All sera were inactivated at 56'C for 30 minutes before use.
Fluorescent Antibody Tests. ;\No:\ and antinuclear
antibody (ANA) were examined by indirect immunofluorescence usinq rat liver sections as described prcviously (16). lmmunoglobulin C was isolated from 3 sera
(patients NSl. O D . and MC). Each was conjuqatrd with
Ruorcscein isothiocyanatc (.%No;\-ronjuqate) (17). Inhibition studies were carried out with 5 to 10 times diluted
ANoA-conjuyatc added to a serial dilution of isolated antigen. After incubation of antiqen hNo.%-conjuqatc mixtures
From the Rheumatic Disease Laboratory, Maine Medi- at 37'C for 2 hours. and refriqeration at 4'C for 2 to 3
days, the mixtures were centrifuqed for 30 minutes at
cal Center. Portland. Xlainc.
12.000 rpm and supernatants were used for dircccimmunoSupported in pan by a qrant from the Maine Chapter of
The Arthritis Foundation.
fluorescent staining.
SHOJI MIYAWAKI, MD: 3rd Department of Internal MediAqar qel diffusion was performed in 0 . 6 2 aqarosc (Scacine, Okavama University Medical School. Okavama. .la- Kem Aqarose. Xlarine Colloids Incorporated, Rockland.
pan; ROBERT F. RITCHIE, MD: Rheumatic Disease Labora- Maine) at p H 7.4.
tor).. Maine Xledical Center.
Isolation of Nuclei(18). Liven were removed fmm
Reprint requests should be addressed to: Roben F.
180- to 220-q albino rats and were immediatelv perf& via
Ritchie, MD.Rheumatic Dixasc Laboratory. Maine ,Medthe portal vein with 20 ml of ice-cold isotonic sodium chloical Center. Portland. XlE04102.
Submitted for publication Xlarch 13, 1973; accepted .July ride. followed by PO ml of 0.25 L1 sucrose ccmaininq 3.3
mM CaCI,. The organs were frccd of excess connrctivc tis18.1973.
726
Arthritis and Rheumatism, Vol. 16, No. 6 (November-December 1973)
NUCLMLAR ANTIGEN
Tdl. 1. S p t x t r o p h o t o m e t r i c Analysis of Nuclear
and Nucltmlar Extracts
Wavelength (nM)
Extracts
Ratio:
2601280
Ratio:
2601235
1.85
1.70
1.20
1.46
1.60
1.50
1.75
' 1.90
1.10
1.35
1.45
0.95
1.63
P~wIoI-No
Phenol-N
CHA-No
trisao
SNoP
Tris-N
SNP
2.20
sue, filtered through 4 l a y m of chmecloth, and then homogenized in 2.2 M sucroseconraining 3.3 mM CaCI2 with
a T d b n pestle in a glass [email protected] (0.006-inch pestle
clearance). T h e homogcnatc was ccntrifuqcd at 4 0 , W for
~
60 m i n u t a at 4' C to sediment the nuclei. T h e pellet was
resuspended in 1.O Sl sucmse containins I.O mM CaCI,
and centrifuged at 3.000s for 5 minute. T h e pellet consisted of highly purified nuclei.
Irolathn of Nucleoli. Nucleoli from rat liver was isolated by a method employing sonic disruption as described
by Murarnarsu ct al(19). T h e nuclear pellets recovered as
described above were resuspended in 0.25 Sl sucrose containinq 3.3 m Y CaCI, (1 ml/g original wciqht of tissue)
and sonicated for 90 to 120 seconds in a Raytheon Sonic Oscillator ( I .O-1.1 amp of ourput current) at 2. C until the nuclei were destroyed as determined by microscopic examination of the fluid after stnining with Azur C. Twentv
milliliters of the sonicated suspension were layered over 20
ml of cold 0.8 to I .O Sl sucrose, centrifuqcd st 2000e for 20
minuta at 4* C (20).The sedimentcd nucleoli were washed
once by suspension in 0.25 Sf sucrose and centrifuqed at
1200s for 10 minuta. This preparation of isolated nucleoli
contained very few nuclei and little debris.
Phenol Exfrucfion (21.22) of Nuclei (Phenol-LV)
and Nucleoli (Phenol-No). Purified nuclei or nucleoli
w e n homogenized in 10 ml of a solution containinq 0.3%
sodium dodccvlsulfate (SDS)(Fisher Scientific Cornpanv),
0.14 M NaCI. 20 &rnl of polyvinylsulfate (PVS) (Sigma
Chemical Company) and 0.05 M acetate buffer p H 5. I .
Homogenization was continued for 1 minute with a Teflon
pestle. An equal volume of 90% phenol contsininq 0.1708hydroxyquinolinc (Fisher Scientific Companv) previously
saturated with the same buffer was added and thr sample
was homogenized for an additional 60 s m n d s . T h e mixture
Table 2. S u m m a r y of Precipitating Reactions in Gels
Antigens
ANA titer and
Patients
NM
ER
CL
SE
SR
MG
OD
DJ
s)
ow
BG
CM
LO
FB
JA
SB
staining pattern
1:64 H + No, 1:500.000 No
1:9OOO No
1:64Sp+No. 1:512 No
1:2048 No
1:64 H + No. 1256 No
1 : 8 S p + N o . 1:128No
1:8OOO No
1:128 H+No, 1:2048 No
1:32 H + No. 1:2048 No
1:32 S p + N o . 1:9OOO No
1% H+ No. 1:2048 No
1:16 H+No. 1:128 No
1:8 S p + No. 1:6400 No
1:128H+No. 1:512 No
1:8OOO No
1:32 H+No. 1:1024 No
Phenol-No*
SS-DNAf
DS-DNAt
Poly 1 4
Poly A-U
+
+
+
+
+
+
+
+
+
+
+
+
--
+
+
+
+
+
+
+
+
+
-
+
-
+
-
'-
+
-
+
-
--
-
-
-
+
+
+
+
+
--
--
-
+
+
-
-
H = homogeneous. S p = speckled. No = nucleolar.
' k i p i t i n line to nucleolar RNA
Wrecipitin line to SS-DNA in Phenol-No a n d f r o m calf t h y m u s DNA
#Fromcalf t h y m u s
M
r and R h e u m a t i s m , Vol. 16,No. 6 (November-December 1973)
727
MIYAWAKI C RlTCHlE
added to the nuclear and nuclmlar residues, homogenized.
and stirred viqomusly nverniqht at 4'C. The suspension
was then ccntrifuqed at 2O.OOQtfor 30 minutes. The supcrnatant was adiusted to dilute salinc-citrate hufler (0.015 M
Nucleolar
NaCI, 0.0015 M sodium citrate) bv qradicnt dialysis aci mmunoPretreatment of tissue
cording to the procedure of Huanq el a1 (24). T h e soluble
section
fluorescence
extract was used as soluble nuclear (SNP) o r nuclcolar p'otein (SNoP).
PBS (0.01 M. pH 7.2
The materials recovered as dwrihcd above were anr0.14 M NaCI)
Present
lyzed spectmphotometrically. T h e 260:2RO and 260/235
0.14 M NaCl
Present
ratio of t h e x materials are summarized in Table 1 .
0.5 M NaCl
Trace
Ofher Antigens. Cytoplasmic ribosomes were prepared
1.0-2.0 M NaCl
Absent
from rat liver according to the method of Schur el a1 ( 9) .
SDC* or CHAt solution (>0.2%)
Absent
T h e phenol extract of these ribosomes was prepared as described above.
*Sodium deoxycholate
Svnrhair polvribonwleotides employed were Pol? JC.
Sholic acid
Poly A-U (Miles Laboratnrv Inc.). DS-DNA. and yeast
RNA (Wonhingon Biochemical Corporation). SS-DNA
was prepared bv heatinq DS-DNAat lO(PCfor 10 minutes
and prompt coolinq in ice. T h e concentrations of thew antiwas shaken vigorously at room temperature for 30 minutes.
T h e multant mixture was centrifuged at 20.000,~for 10 %ens were: SS-DNA at 500 &mi. DS-DNA and yeast
RNA at 1 mq/ml. and polyrihnnuclcntides at 100 &ml.
minutes. T h e aqueous phase was transferred to another
En:ymes. Bovine pancreatic ribonuclcasc (RNase).
centrifuge tube and a fresh volume of the phenol solution
dcsoxyribnnucleasc I (DNasc). and tppsin (two times cryswas added. After shaking for 10 minutes and funher centritallized) were obtained from Wonhinqton Biochemical
fugation as described above, the aqueous layer was reCnrporation.
moved. To the water soluble fraction, 2 to 2 %volumes of
Sucrose Densify Grodienf Sedimentation (22) and
ethanol containing 2% pntassium acetate were added and
Ukracentnjugal.4nalysis. One milliqram of Phenol-N or
allowed to precipitate overnight at - 2 P C. T h e precipitates
Phenol-So was dissolved in a small amount of 0.02 11 snwere washed once with 75% ethanol. dissolved in a smnll
volume of 0.05 hf Tris-HC1 buffer p H 7.5. and used as dium acetate buffer. pH 3.1 containinq 0 . 1 &I NaCl. 1.0
mM EDTA. and 20 rqlml of PVS. and layered on 13 ml of
Phenol-N or Phenol-No. These materials were analyzed
s ~ r o p h o t o m e t r i c a l l yat wavclenghs of 235.260. and 280 a 10 to 40% linear qradient of a sucrose solution containing
the same compnncnts. T h e gradient was c c n t r i f u d in a
nM .
Cholic Acid Exfracfionof Nucleoli (CH.4-XO). A Spincn S W 40 Ti rotor at 39,000 rpm for 17 hours at 5.C.
modification of the method of Birnstiel et ul was used (23). Fractions were colln~edfrom tuhes and analyzed at 260
nM spcrtrophotometricall~.Fractions under the same optiNuclcolar fractions were resuspended in Tris-HCl buffer
cal
density were pooled separately and precipitated with 2
(pH 7.6) containinq 2 mM MgCl, and homogenized with
an q u a 1 volume of 1% cholic acid in the same buffer. T h e to 2 5 volumes of ethanol containinq 2% potassium acwate.
homocenate was diluted with 2-3 volumes of water and The. precipitates were dissolved in 0.05 L.1 Tris-HC1 buffer,
crntrifuqed for 20 minutes at ~0.ooO.q.Thesupernatant was p H 1.5.
Analytical ultracentrifuqation was done in a Beckman
removed, dialyzed. against 0.05 51 Tris-HCl huffer ( p H
,Model E with an ultraviolet photoelectric scanner (LW:
7.5) for 3 days. concentrated. and then used as CHh-No.
Saline Exfrocfwn (21) of Nuclei and Nucleoli. Tris- 254 n51). Fiftv micrograms of material were dissnlved in
NaCl extract was obtained by treatment of the isolated nu- 0.9 M NaCI and 0.01 M sodium citrate and centrhqed at
clei or nucleoli as described above with 2 extractions in 0.11 31.410 rpm.
M NaCl containinq 0.05 Tris-HCl buffer at p H 7.5 and 0.2
mg/ml of PVS. T h e nuclei or nucleoli were homoqenizcd in
RESULTS
this solution for 1 minute and centrifuqed at 4 0 0 0 ~for I0
Patients
Serum
(Table
2)
minutes. T h e supernatant was dialyzed against Tris-NaCl
buffer for 3 days to remove PVS. (Inasmuch as PVS proSixteen sera from patients with high .\Noh
duced a prccipitin line indistinguishable from a classic anti- titers frequently had clinical diaqnoses of sssgen-antibody reaction. extensive dialysis is required for
temic sclerosis o r Raynaud's phenomenon as reproper evaluation.) This material was used as thc TrisNaCl extract of nuclei (Tris-N) o r nucleoli (Tris-No). After ported elsewhere (25). Four patients had only
several washings with Tris-NaCI buffer, 2 51 NaCl was antinucleolar antibody fluorescent patterns at
Table 3. Effect of Aqueous Buffer) and Detergent
Solution on Nucleolar l m m u n o f l u o r e s c e n t Patterns
720
Mhritir and Rheumatism. Vol. 16, No. 6 ( N o v e m k - D m m b r 1973)
.-
Fig 1 Precipitin reaction f two s rum antinucle lar antibodi 5 with various soluble antigens. (A) Serum SE containing only ANoA reactivity with phenol nucleolar extract. 1) SS-DNA
calf thymus. 2) Phenol-No. 3) DS-DNA (calf thymus). ( 8 ) Serum NM containing an extremely
high titer of ANoA and only traces of other antibodies produces additional precipitin lines with
other antigens. 1) Poly IC. 2) Phenol-No. 3) SSDNA (calf thymus).
Fig 2. Precipitin reaction of 3 ANoA positive sera (SA,LO. OD) with single-strand calf thymus
DNA (SS-DNA).
&Uuik and Rheumotiun, Vd. 16, No. 6 (Nowmber-Dwemkr 1973)
729
MIYAWAKI & RFTaaE
Fig3. Precipitin reaction
of an ANoA positive xrum
(OD) with various soluble
antigens. A line of identity
is seen with all materials
indicating the prcscna of
SS-DNA in each. 1) Tris-N.
2) SS-DNA. 3) CHAHo.
4) SNOP. 5) Tris-No. 6)
SNP.
all dilutions, whereas, the remainder possessed
antibodies to other portions of the nucleus. The
predominant antibody in each patient was
against the nucleolus as demonstrated by serial
dilution and fluorescent antibody testing.
Removalof Nucleolar Antigen From
Nuclear Substrates
The fixed liver sections were pretreated with
a variety of aqueous buffers and detergents for
30 minutes before incubation with an ANoA
positive sera. As illustrated in Table 3, the
nucleolar antigen(s) were extracted by pretreatment with solutions containing more than
0.5 M NaCl. or 0.2% sodium deoxycholate or
cholic acid solutions.
730
Identificationof Nucleolar Antigen
(Table 2)
The precipitin reactions produced in Ouchterlony diffusion gave 1 or 2 t i n e between
ANoA positive sera and Phenol-No as illustrated in Figure 1. The first sharp precipitin
line close to the antigen well was common to all
ANoA positive sera. The second line, broad and
less discrete, usually located midway between
the antigen and antibody wells, was found in 6
ANo.4 positi;e x r a : two with only ANoA and 4
with mixed immunofluorescent patterns. T h e
six sera gave a single line with SS-DNA ppared from calf thymus (Figure 2) that fused
completely with the second line produced by
Phenol-No (Figure 1 B).
ArthriUr and Rheumatism, Vol. 16,Mo. 6 ( N o w n k r 9 . a n r k r U 9 3 )
NUCLEOLAR ANTIGEN
.,... ...
a
Fig4. Precipitin reaction of two sera containing ANoA: serum SE containing only antinucleolar
antibody: serum SLE from a patient with systemic lupus erythematosus containing antinuclear
antibodies(A) (left) RNase treated Phenol-No (1) produced no band while the same material
treated with DNase or trypsin produced a single sharp band close to the antigen well. (B) (right)
DNase treated Phenol-No ( 2 ) produced no band while the same material treated with RNase and
trypsin produced a single diffuse band closer to the antibody well. 1) Phenol-No treated with
RNase. 2) Phenol-No treated with DNase. 3) Phenol-No treated with trypsin.
In addition, a precipitin line between SSDNA and the serum from a patient with SLE
produced with Phenol-No (Figure 1 B).
was identical. It seemed likely. therefore, that
Effect of Enzyme on Extracts
After dialysis against 0.01 %lTris-NaCI buffer at pH 7.2 containing 2 mXI 51gCI2,PhenolN o was incubated with proteolytic and nucleolytic enzyme (final concentration of enzyme
0.4 mg/ml) for 1 hour at 37" C.
The antigen which produced the first line
was completely destroyed by digestion with
RNase. DNase and trypsin had no effect on this
antigen (Figure 4 A). The second line. identified as SS-DNA, as described above was sensitive only to the digestion with DNase (Figure -4
B). The antigen in other nuclear or nucleolar
extracts that reacted with ANoA sera was dip t e d with DNase. All antigens were insensitive to periodate oxidation.
It appeared, therefore, that the nucleolar
phenol extract contained two antielens: nucleolar RNA and DNA.
the second precipitin line was produced by SS-
DNA.
Only 6 sera that reacted with SS-DNA gave
one line against Phenol-N, CHA-No, Tris-N,
Tris-No, SNP, and SNoP, and all were identified as having been produced by SS-DNA (Figure 3). None of the above extracts produced the
first sharp precipitin line observed when ANoA
positive sera were reacted with Phenol-No. Material isolated from cytoplasmic ribosomes,
ribosomal RNA, yeast RNA, and DS-DNA
from calf thymus did not react with ANoA positive sera.
Precipitating antibodies to poly I-C were
found in the sera of 7 patients. of which 6 reacted with SS-DNA and 3 with poly A-U. All
precipitin lines produced spurs over one another and were not identical with the first line
Arthritis and Rheumatism, Vol. 16, No. 6 (November-December 1973)
731
Cohn
weights. Although Phenol-N was she.. .
have a very similar sedimentation pattern ;i
Phenol-So, only SS-DNA antigen was detectable; the RNA related antigen being absent.
FrIt
Ultracentrifugal Analysis
T h e fractions that contained nucleolar RNA
that reacted with ANo;l sera were collected
from a sucrose density gradient and its sedimcntation coefficient measured by ultraviolet photoelectric scanning. T h e sedimentation coefficient
was calculated at 6.8S,which agreed with the S
value postulated from the pattern produced ,in
the sucrose density gradient.
0
p 1.0
Phenol-No
,- Nucleolor Antigen
(RW
I
U
5
10
15
20
25
50
Fig 5. Sucrose density-gradient patterns of Cohn
Fraction II. Phenol-No and Phenol-N.
Sucrose Density Gradient Sedimentation
Analysis
Figure 5 shows the sucrose density gradient
sedimentation pattern of Phenol-So and Phenol-N as compared with Cohn fraction 11. Two.
major sedimentation classes were seen, both in
Phenol-No and Phenol-S. One was a large
amount of material with an approximated sedimentation coefficient of 4 to 7s and the balance
of the material with an S value of greater than
18. When these fractions were checked by immunodiffusion against A S o A positive sera the
nucleolar RS=\ antiqen was located only in the
Phenol-No fractions of approximately 7%
whereas DN.1 fractions that reacted with
AXoA sera were found in the reqion from 4s to
over 18s. This suqqested that the extracted
DNA, while still immunoreactive, had been degraded to molecules of varying molecular
Adsorption Study
Nucleolar immunofluorescent staininq was
almost completely inhibited by adsorption of
ANoA-conjugate with Phenol-No (Table 4).
When Phenol-No was digested with R N w before incubation with ANo;Z-conjugate the inhibitinq ability of this material was markedly
diminished. N o inhibition of nuclmlar bindinq
was observed when Phenol-No was digested
with both RNase and DNase. Prior incubation
of AKoA-conjugate with SS-DNA from calf
thvrnus. Phenol-N. CHA-So. Tris-N, Tris-No.
Table 4. Adsorption of Fluorescinated Human
ANoA Conjugate with Various Antigens
ANoA conjugate
absorbed
Phenol-No
Phenol-N
CHA-NO
Tris-No
Tris-N
SNP
SNoP
SS-DNA
DS-DNA
Ribosomes
Poly 1-c
POIYA-U
Yeast RNA
Nucleolar imrnunofluorexence
Almost completely inhibited
Slightly inhibited
Slightly inhibited
Slightly inhibited
Slightly inhibited
Slightly inhibited
Slightly inhibited
Slightly ihnibited
Not inhibited
Not inhibited
Strongly inhibited
Slightly inhibited
Not inhibited
NUCLEOLAR ANTiGEN
Table 5. Incidenceof PrecipitatingAntibody to
Nuclrolar RNA in Patients With Other Rheumatic
Diseases
~~~~~
~
Diseases
~~
Patient No.*
Positive No.
SLE
36
Pronestyl lupus
12
17
9 (25%)
4 (33% )
6 (35%)
RA
*All
positive ANA ( > 1:8)
SNP, and SNoP only slightly inhibited nucleolar staining. However, after digestion of these
antigens with DKase, no inhibition was observed. Poly I-C adsorbed ANoA strongly and
p l y A-U weakly. In contrast, incubation of
ANoA-conjugates with DS-DNA, cytoplasmic
ribosomes, ribosomal RN.4, yeast RNA, even in
large excess, had no effect on ANoA binding.
PrecipitatingAntibodies to Nucleolar
RNA in Other Diseases
The results of a study of the serum of patients
with SLE,pronestyl-lupus. and rheumatoid arthritis for antibodies to nucleolar RNA is illustrated in Table 5. Precipitating antibodies to
nucleolar RNA were found in 9 patients with
SLE (25%), 4 with pronestyl-lupus (33%).and
6 with RA (35%). Among these positive sera, 7
gave precipitin reactions with DNA present in
the Phenol-No, 3 had systemic lupus erythematosus, 1 pronestyl-lupus, and 3 with RA. All
sera had a positive study for antinuclear antibodies, however, ANo.4 did not predominate.
Several other immunofluorescent patterns were
Seen on testing these same sera.
DISCUSSION
The present study sugsests that the major
nucleolar antigen is nucleolar-RNA with a sedimentation coefficient of 7 s . This conclusion was
based on four major findings: 1) The antiqen
was found in an extract of purified nucleoli. 2)
It was not extractable from whole nuclei or the
cytoplasmic fraction. 3) Nucleolar immuno-
fluorescence was inhibited by absorption of
ANoA positive sera with this antiqen. 4) It was
sensitive only to digestion with RNase.
Recent studies on nucleolar RNA (26,27) reveal that there are 4 major sedimentation
classes of nucleolar RNA with approximate
sedimentation coefficients of 4 to 7 s . 28S, 3 3 .
and 45s. Among the rapidlv sedimentinq forms
of RNA those with sedimentation coefficients of
more than 28s have been identified as the precursors of ribosomal RNA. In contrast, slowly
sedimenting forms of 4s-7s have been thought
to be localized primarily in the nucleolus. Their
very slow turnover rates and nucleotide composition distinguish them from the hiqh molecular
weight forms of RNA. While their functions at
present are not known, they are presumed to be
part of either the svnthetic or control mechanisms of the nucleolus and nucleus. Our findings identifying ANoA as reactinq with 7s nucleolar RNA has 2 distinct implications: It
provides a means of identifying the presence of
the unique low molecular weight RN.\ in nucleoli
immunochemical means and supports
the specificity of ANoA as observed by immunofluorescence in their reaction only with cellular
nucleoli.
The data also indicates that DNA present in
the nucleolar phenol extract was reactable with
ANoA though its reactivity was weaker than
that of nucleolar-RNA. As compared with nucleolar-RNA, the origin of DNA in the phenol
extracts is unclear, since DNA reacting with
ANoA could be extracted not only from nucleolar fractions but from whole nuclear fractions
and calf thymus. However, as nearly one-third
of the cases with positive serum ANoA-even
samples containing only antinucleolar bindinq
specificity-were capable of reactinq with SSDNA, we cannoi exclude the possibility that
DNA associated with nucleoli, presumablv
perinucleolar nucleolus-associated DNA. and/
or intranucleolar DNA, might serve as ANoA
antigen independently of or in connection with
nuclear DNA. .Moreover, the fact that our
ANoA which reacted with SS-DNA also re-
Arthritis and Rheumatism, Vol. 16, No. 6 (November-December 1973)
733
MlYAWAKl L RltCHIE
acted with synthetic RNA. including poly I-C
and poly A-U, might provide further evidence
of antigenicity of DS.4 because, as reported by
Koffler et a1 (28) and Natali and Tan (19). the
antibodies reactive with polyribonucleotides
might have a greater affinity for DN.4. The
ability of Poly I-C to inhibit nucleolar staining
was unexpectedly strong in our absorption experiment. Since the Poly I-C precipitin system
appeared to be different from that of the nucleolar 7 s RNA precipitin, the strong inhibiting
ability of Poly I-C may be due not only to the
cross-reaction of SS-DNA antibody as mentioned above but also to a nonspecific coprecipitation of the antibody.
Using immunofluorescence we determined
that nucleolar antigens were extractable by the
pretreatment of tissue sections with some aqueous buffers and detergents. Application of these
observations suggested that extraction of whole
tissue with such solutions should have resulted
in the removal of 7 s RNA. The failure to do so
may be explained on the resistance of the perinucleolar chromatin shtll maintained by the
divalent calcium ion necessary for maintaining;
the shape of nucleoli and nuclei during separation (19). Extraction in the presence of SDS and
phenol has made it possible to extract an immunoreactive nucleolar 7s RNA and DN;\. However, we cannot exclude the possibility that nucleolar antigen is actually present in our other
isolates but at concentrations too low to be detected by the method employed. It is possible
that, because of the protein denaturant qualities
of these chemicals, nucleolar protein that makes
up approximately 80% of the nucleolar dry
weight (19,27) may have been destroyed.
The RN.4 system relating to human serum
ANoA described here seems to be unique and
different from RNA systems reported by others.
Watanabe et a1 (30) reported 2 sera from patients with SLE that contained antibodies reacting with nucleoli of mononuclear cells. Since
these antibodies also reacted with cytoplasm
and were absorbed with cytoplasmic ribosomes
(not found in our cases), it is likely that they ob734
served different and more rapidly sedimenting
RNA systems, probably more closely related to
that of ribosomes as described by Stugill and
[email protected]) and Shur et a l ( 9 ) . M o r e r
e
cently, antibodies to a nuclear RNA system
have been reported by Mattioli and Re&
lin (12) to be present in the sera of some
patients with SLE and by Sharp et af (13) in
patients with an apparently distinct rheumatic
syndrome. In the latter, clinical features of a
mixed connective tissue disease manifested by
Raynaud's phenomenon, tight skin, anhralgia,
myalgia. etc., are similar to those of our cases.
However, the antiqen identified in both reports
indicated a ribonucleoprotein was apparently
responsible for a speckled imrnunofluomscmt
pattern quite different from the cases reported
here.
The antibody to 7 s nucleolar RNA was also
found in some of our patients with SLE, pronestyl lupus, and RA. In these patients the
staining pattern could not be interpreted as
being predominantlv of the nucleolus becaused
the interfering; fluorescence of surrounding nuclear structures. Nevertheless, the fact that
ANoA specific for 7 s nucleolar RNA could be
demonstrated in about one-third of the patients
with rheumatic diseases when precipitin rcactions are emploved suqgests that ANoA m a y not
be an unusual antibody among systemic rheumatic diseases. The differentiation of thew various individual antibodies has been thought in
the past to be of diagnostic significance and perhaps to offer a clue to the pathqenesis of these
diseases. A possible protective role for A N 4
similar to ENA antibody (31) could be polated since our patients who had ANoh in t h e r
sera usually have a prolonged and mild clinical
course without renal manifestations and a q w d
response to corticosteroid therapy.
ACKNOWLEDGMENTS
We would like to thank Dt. McDonald G m m d&c
University of New Hampshire for assistance in ulasar-
trifuqal analvsis, The Department of ,Medical Phamgraphy for preparing the illustrati(~u.and .Mary K i d d l
for secretarial assistance
Arthritis and Rheumatism, Vol. 16, No. 6 (Novembw-D8cember 1913)
NUCLEOUR ANTIGEN
tive with SLE sera. J Immunol 107:1 8 1-1290,
1971
\[ixed
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thioacetamide on the composition ant1 Iiiosvnuble nuclear antigen precipitatins with sera of
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MlYAWAKl 6 RlTCHP
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K: T h e Nucleolus. N e w
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736
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Arthritis and Rheum8tirm. V d . 16, No. 6 (November-December 1973)
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