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Specificity and reactivity of cytoplasmic and nucleolar antibody in SLE sera.

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Specificity and Reactivity of Cytoplasmic and
Nucleolar Antibody in SLE Sera
By NOBUOWATANABE,
M.D., HERBERT
M. FISHER,M.D., AND WALLACE
V. EPSTEIN,
M.D.
The serum of 2 of 31 patients with SLE
was found to contain an antibody to a
previously unrecognized RNA-containing
antigen found in human mononuclear cell
cytoplasm and nucleolus. When tissue
fixed, this antibody proved capable of
H
directed toward cytoplasmic determinants have been
identified in the serum of patients with
lupus erythematosus, as well as those with
thyroiditis, biliary cirrhosis, and several
other diseases of equally uncertain etiology.
Microsoma1,l ribo~omal,~.~
lysosomal,4 and
mitochondria1 antibodies,1.6 as well as antibodies directed toward less well-defined
substructures,8 have been described by
means of various serological technics including complement fixation, bentonite flocculation, and precipitation. Cytoplasmic
antibody has also been studied by means
of fluorescent antibody technic^.^'^ Although less sensitive than the complement
fixation procedure, fluorescence does allow
visual localization of the site of the antibody attachment and may define the sequence of globulin fixation to tissue sites
when multiple antibodies are present. In
the course of examining the sera of patients
with SLE for antinuclear antibody, we
found that 9 of 31 sera demonstrated IgG
UMAN ANTIBODIES
From the Rheumatic Disease Group, Department of Medicine, University of California, San
Francisco Medical Center, San Francisco, California.
This study was supported b y Grants 1229 and
2 A 5 1 2 4 , National Institutes of Health, Bethesda,
Maryland.
Presented in part, at the Fourth Pan-American
Congress of Rheumatology, Mexico City, Mexico,
October 22-26, 1967.
ARTHRITISAND RHEUMATISM,
VOL. 12, No. 3
fixing the patient's own rheumatoid factor and human complement components.
No recognizable inhibition of complement
fixation by rheumatoid factor could be
demonstrated by the fluorescent antibody technic.
antibody to mononuclear cell cytoplasmic
elements. Two of these demonstrated IgG
antibody directed to the nucleolar structure
as well, and these were studied in detail.
MATERIALS
AND METHODS
Fluorescent antibody technic. The multilayer
method of Weller and Coons8 was employed.
The source of fluorescein-labeled antisera to human IgG, IgM, and complement (PIC) was of
commercial origin ( Hyland Laboratories, Los
Angeles). Normal pooled IgG (Cohn fraction 11,
Squibb lot 2001 ) linked to fluorescein isothiocyanateg was used as the reagent for the detection of
tissue-fixed rheumatoid factor. This conjugate was
aggregated by heating it in a water bath to 66OC
for 20 min. just prior to its application to the cell
preparations. The tissue substrate consisted of human peripheral white blood cells obtained from
pooled whole venous blood in citrate anticoagulant
and sections of normal human thyroid tissue cut
in a cryostat. The buffy coat was diluted in normal saline and fixed to glass slides by means of a
cytocentrifuge (Shandon Scientific Co. Ltd., London). Serum was applied undiluted to the substrates. A Zeiss binocular ultraviolet microscope
using an Osram HBW-2OOW light source and
NOBUOWATANABE,
M.D.: Exchange Fellow of
Japan and American Rheumatism Associations.
HERBERT
M. FISHER,M.D.: Trainee, National Institutes of Health. WALLACEV. EPSTEIN,M.D.:
Associate Professor of Medicine, University of
California Medical Center, Son Francisco, California 94122.
Reprint requests should be addressed to Dr.
Epstein.
(JUNE 1969)
173
174
WATANABE, FISHW,
UG2 excitor filter was used for observations.
S m r n fractionation. Serum was passed through
a Sephadex GLBBlO column. Two fractions were
obtained, the h t rich in IgM and the second
rich in I&.
InhibbHton of wra. An aliquot of whole serum
was treated with yeast RNA (Sigma Chemical
CO.) or with RNA isolaterl from rat liver ribosomes
(generously provided by Dr. Peter Schur) 5.0
mg./ml. at 37' for 1 hour. A similar specimen
was mated with calf thymus DNA, 5.0 mg./ml.
~Nutrltional Biochemical Corn.) under the same
conditions.
Source of complement. Undiluted norma1 human whoIe serum stored at -7OOC was the snurce
of homologws complement. An aliquot of patient's
serum was stored at -70% to preserve autologous
complement activity. The serum hemolytic cornplement content was determined by the m e t b d of
_
N & d h ( $ I g )
-
++
I
Kabat and Mayer.11
Test for rheumatoid factor a d i v f t y . The hmnagglutination methd of Hellerl2 WM employed,
using sheep red cells mated with human Cohn
fraction 11.
RE~ULTS
Table l.--Staioing Pattern Using
Flumesa?nt Anti-IgG
m mar
<l:POO
1:m1:B(Oo
>1:8100
Na ot
17
8
3
could b e detected in the sera of 9 of 31
patients having SLE.These same nine sera
fixed IgC to the cytoplnsm of thyroid epithelial cells. Two of these nine also reacted
with constituents of the nucleolar structure.
We have attempted to define something of
the nature of this antibody system by a detailed study of the two sera that showed
both cytoplasmic and nucleolar antibody.
The prevalence of cytoplasmic antibody in
the sera of patients with diseases other
than SLE has not been examined.
Appliation of either of these sera to a
substrate of normal peripheral human blood
leukmytes revealed IgG fixation to the cytoplasm and nucleoli of mononuclear cells but
not to the cytoplasm of plymorphonuclear
leukocytes (Fig. 1). There was very slight
fixationof I g C antinuclear antibody to neutrophil but not mononuclear cell nuclei in
the serum of patient P.H. Addition of rat
liver rnicrosomal RNA or yeast RNA (0.1
cc. of a 5.0 mg./rnl. solution to 0.1 cc. of
whole sentm), but not calf thymus DNA,
in the same concentration, almost cornpletely removed the ability of both sera to
6x IgG to thc cytoplasmic and nucleolar
The sera of 31 patients with SLE were
screened for cytoplasmic antibdies (Table
1). Twenty-two reveaIed no staining of
either the cytoplasm or nucleolus of the
substrate cells with fluoresceinconjugated
anti-IgC. Application of the other antiserum
conjugates after any of these 2!2 sera also
failed to demonstrate staining except for a
small number of polymorphonuclear leukocytes that showed intense fluorescence
of large cytoplasmic granules after antiIgM or of conjugated normal human IgC.
Counterstnining reveals these brightly
staining granules were within eosinophles.
Nine patients demonstrated positive cytoplasmic staining in mononuclear cells but
not in polymorphonuclear ceIS. Two of
thae nine manifested nucleolur fluorescence as well. Twenty-eight of these sera
were teskd for rheumatoid factor activity
by the FII hemagglutination test. The sites.
range of titers is shown in Table 2. TwentyApplication of a fluorescein-labeled anthree showed antinuclear antibody. There- tiserum to human IgM after either of these
fore, antibody to cytop?asmic determinants two SLE sern caused cytoplasmic staining;
CYTOPLASMIC AND NUCLEOLAR ANTIBODY I N SLE SERA
175
Fig. 1.-Normal human peripheral blood leukocytes treated with patient’s serum and
then fluorescein-labeledantiserum to IgG, showing strong mononuclear cell cytoplasmic
and nucleolar staining. X 1000 (originalsize reduced).
Fig. 2.-Same substrate as in Fig. 1, treated with patient’s serum and then fluoresceinlabeled antiserum to human IgM. X 1000 (original size reduced).
no fixation of IgM globulin to nuclear or they had been exposed to either of these
nucleolar sites was demonstrated (Fig. 2). two sera. The fixation, in contrast to that
Further information concerning the nature shown by the antisera to IgG and IgM, was
of the IgM antibody contained in these two punctate and granular rather than homosera was provided by application of fluores- geneous (Fig. 3).
cein-labeled heat-aggregated normal huTo clarify the sequence of events proman IgG. This reagent was fixed only to duced by the application of these sera to
the cytoplasm of mononuclear cells after normal leukocytes, chromatographic frac-
pis. 3subrtrata aa in Fig. 1, heated with patient's serum and than fluh k d normal human 1%: heatel to 63-C for 15 minutes. X 400 {ariginal& reduced).
the sera were used. The IgEridl tim activity at the concentrations used for
fndon!? of these 8erB were applied to the the fluomicent antibody studies.
tions of
leukocyte substrate. Labeled antI-IgC revealed sit= of cytoplawnic and nucleolar
fixation, as desmibed before when the
W h l e serum was d No SEaining was
noted when labeled anti-IgM or heatm t e d
X@ WBS 4.
Application of the IgM-rich fraction alone failed to
d e p i t globulin on any cellular site mi determined by any of the described fluore9~eh-bkledmgmh. Ap~liration Of the
IgGrich fraction and then of the IgM-rich
fracth resulted in a pattern ofcytoplasmic
staining identical to that of the whole
sem,indicating that the sequence of h a t
h to cytoplasmic sites involves fint the
IgG d only tben of IgM mbl-IgC:globulin. Tbis IgM apparently bas &dent unbwnd m p t o r sites to bind the fluotesceinlabeled heat-aggregated n o d I@. The
IgGzich fmctions of the patient's serum at
the concentrations used had no signihnt
antIglobulln activity, while the isolated
IgM fraction demonstrated hemagglutina-
Application of fluorescent-hbkd -tiserum to human pic c o m p b t c o m p
nent after application of the test sera wbicb
had been stored at -7OOC d t e d in stahing of mononuclear cell cytoplasm and
nuchlus. The complement amtent of
serum P.H. at this time was 95 C'Hm units
per milliliter of m (aormal, 2&4!5
units), and of serum LL. was 8 1 C b
units. A similar cytoplasmic and nudeoh
staining pattern for sites of complement
&ration could be produced by applieabIon
of the IgC-ricb ftaetion but not the IgMrich fraction prior to rhe application of a
fresh normal Serum which acted as the
source of mmplement (Fig. 4).
W e attempted to demonstrate competition betwen rtaeumatoid factor and buman
mplement m p o n e n t s for the same h
on the IgG Gred to cytoplasmic a d nuclew
lar antigen. No matter what the qtlemm
of the addition of IgM-rich fmcdon ob of
complement source, flwmscent-labeled an-
CYTOPLASMIC AND NUCLEOLAR ANTIBODY IN SLE SERA
177
Fig. 4.--Same substrate as in Fig. 1-3, exposed to patient's serum, then homologous
complement source, and then fluorescein-labeled antiserum to human Pic. X 1000
(original size reduced).
tisera showed that both rheumatoid factor
and PIc complement became attached to
the IgG cytoplasmic antibody. We could
not recognize a diminution in the intensity
of staining for PICwhen IgM rheumatoid
factor was allowed to fix to the same site.
DISCUSSION
Antibodies to cytoplasmic constituents in
the serum of patients with systemic lupus
erythematosus have been reported by several investigators. Mitochondrial, microsomal, and soluble cellular fractions have all
been found to react with SLE sera by bentonite flocculation and by complement fixat i ~ n . Miescher
~,~
et al.13 and Wiedermann
et al.4 have described antilysosomal activity
in patients with SLE and hepatitis using
the same technics. A ribosomal fraction has
been obtained by Dausset14 from human
leukocytes and by Sturgill and Carpenter2
from rat or rabbit liver. Antiribosomal activity was demonstrated by this investiga-
tion with antiglobulin consumption and
bentonite flocculation respectively in SLE
and other conditions. More recently, Schur
et a1.3 have purified mammalian cell ribosomes and by immunoprecipitation have
found ribosome antibody in 11 of 85 patients with lupus nephritis. Finally, Rumke
and Buter15 detected mitochondria1 antibodies in patients with primary biliary cirrohosis employing the fluorescent antibody
technic. It is certainly not clear at present
just how many antibody systems for cytoplasmic determinants are to be found in
association with SLE and other human diseases. It is even less clear with respect to
nucleolar antibody. In the present study,
two of the nine sera containing antibody
to mononuclear cell cytoplasm also caused
nucleolar fluorescence. These studies are
consistent with either an additional antibody system present in these two sera or
differences in the concentration of an RNA
antibody, with penetration to the nucleolus
178
related to concentration. Antibody directed
to the nucleolus has not been reported associated with anticytoplasmic activity in
SLE, although nucleolar antibody has been
recognized in patients with various connective tissue diseases.lG
Although some of these workers have
used the fluorescent antibody technic with
a human leukocyte substrate, no note was
made of the staining of mononuclear without staining of polymorphonuclear leukocyte cytoplasm. The two SLE sera of the
present study that de.monstrate both cytoplasmic and nucleolar antibody appear to
have specificity for an RNA-containing
moiety of mononuclear cell nucleoli and
cytoplasm. Cline,17 in a detailed review of
the metabolism of circulating leukocytes,
describes numerous differences in the enzyme and metabolic systems of granulated
leukocytes as distinct from mononuclear
cells, but records no RNA-containing complex present in mononuclear cells and absent in the granulated cell cytoplasm. We
have no further knowledge of the antigen
recognized by the IgG globulin of these
patients with SLE.
WATANABE, FISHER, EF’STEIN
while all sera tested with antinuclear antibody of low molecular weight (IgG) reacted with unaltered fresh granulocytes,
only one-third of the sera with IgM antinuclear antibody and only one-half of those
with IgA antinuclear antibody combined
with unaltered granulocytes. To obtain
maximum accessibility, it was found necessary to alter the substrate cells by repeated
freezing and thawing. This may be applicable here to account for the lack of fixation
of the rheumatoid factor to nucleolar-fixed
antibody. Although indirect evidence has
been presented forlo and against*O the
fixation of human rheumatoid factor to
nuclear-antinuclear combinations, the present demonstration using fluorescein-labeled
normal IgG provides direct proof of fixation
of the patient’s own rheumatoid factor to
the IgG cytoplasmic antibody. Employing
the patients’ IgG fractions followed by their
IgM fractions duplicated the pattern demonstrated when whole serum is used and
thus established that the sequence of attachment to cytoplasmic sites involves first
IgG globulin and only then of IgM antiIgG.
At least one component of the autoloBecause these 2 patients had significant
gous
complement system (PIC) of the 2
titers of antiglobulin activity ( rheumatoid
patients
was found to combine at the same
factor), we could study the attachment
mononuclear
cytoplasmic and nucleolar
sequence of various serum components. The
as
the
patients’
IgG. Addition of the
sites
rheumatoid factor activity measured by the
immunoglobulins
and
of the sources of comFII hemagglutination test was confined to
plement
individually
demonstrated that
the IgM-rich fraction, and no IgG rheumasuch
complement
fixation
required the prior
toid factor could be demonstrated. ApplicaPIC component
fixation
of
IgG.
The
same
tion of fluorescent antiserum to IgM after
reacted
similarly
from
a
homologous
comeither of these two sera did stain the monoplement
source
when
added
after
the
panuclear cytoplasm but did not stain nucleoli.
tient’s
IgG.
This may be due to the inability of the
We attempted unsuccessfully to demonlarge IgM molecule to penetrate the nuclear membrane, although it readily fixed strate competition between complement
to the IgG attached to the accessible cyto- components and rheumatoid factor for the
plasmic antigen. The question of the ac- same binding site. Complement attached
cessibility of nuclear sites to molecules of to the IgG fhed on the cytoplasm and
the size of IgM rheumatoid factor has been nucleolus whether or not the patient’s own
examined by Elling,l* who found that, rheumatoid factor was added first. Such
179
CYTOPLASMIC AND NUCLEOLAR ANTIBODY I N SLE SERA
of Results, Sera of P.H. and I.L.
Table 3.-Table
Serum Fraction
Subshate
Whole serum
Poly nuclei
Mono cyto*
Mono nucleoli
Mono cyto
Mono nucleoli
Mono cyto
Mono nucleoli
Mono cyto
Mono nucleoli
Mono cyto
Mono nucleoli
Mono cyto
Mono nucleoli
IgG
IgM
IgG, then IgM
IgG,thenIgM
then C'H $
I&, then C'H
-~
~
FlAnti-I M
P.H.
fL.
P
%
?
'
E
.
+++
++
++
++
+++
+++
++
Fl Anti-&a
IL.
P.H.
Fl N.I S
P.H.
I.L.
I
-
+++
+
-
+++
+
~
Mono cyto = mononuclear cell cytoplasm.
t f= weak staining.
$ C'H = normal human serum as source of complement.
competition has been shown by methods
other than f l u o r e s c e n ~ e ? ~An
- ~ ~ isolated
IgM from a strongly seropositive rheumatoid arthritic serum also failed to prevent
complement fixation in this system. (See
Table 3.)
The place of rheumatoid factor in SLE
remains
but a protective
has
been proposed b y Davis and B01let~~
based
SUMMARY
AND
on clinical-serological correlation. Examination of the kidney of one of the patients
(P.H.) by percutaneous biopsy revealed
the deposition of IgG and IgM globulins as
well as complement components within involved glomeruli.
ACKNOWLEDGMENT
The technical assistance of Mrs. Elaine Hill is
gratefully acknowledged.
CONCLUSIONS
In 9 of 31 SLE sera examined, an IgG antibody with specificity for normal human
mononuclear but not polymorphonuclear cell cytoplasm was detected. Two of these
nine sera also showed an IgG that bound to mononuclear cell nucleoli. Once fixed to
cytoplasm, the IgG antibody can fix both autologous rheumatoid factor and components
of complement. The IgG antibody on the nucleolus can fix complement components but
not rheumatoid factor, possibly due to the inability of rheumatoid factor to penetrate
the nuclear membrane. The fluorescent antibody method has enabled us to establish
the sequence of attachment of IgG, IgM rheumatoid factor and complement to the
antigenic sites. No recognizable inhibition of complement fixation by rheumatoid factor
could be demonstrated by the fluorescent antibody technic.
SUMMARIO
IN INTERLINGUA
Esseva constatate que le sero de 2 de 31 patientes con disseminate lupus erythematose contineva un anticorpore anti un previemente nonrecognoscite antigen0 a contento
de acido ribonucleic presente in le cytoplasma de human cellulas mononucleari e etiam
in le nucleolo. In tests de fixation de tissu, iste anticorpore se monstrava capace de
fixar le factor rheumatoide del patiente mesme e etiam human componentes de complemento. Per medio del technica a anticorpore fluorescente nulle recognoscite inhibition
del fixation de complemento per factor rheumatoide poteva esser demonstrate.
WATANABE, FISHER, EPSTEIN
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