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IgM anti-histone H-3 antibody associated with undifferentiated rheumatic disease syndromes.

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39
-
IgM ANTI-HISTONE H-3 ANTIBODY
ASSOCIATED WITH UNDIFFERENTIATED
RHEUMATIC DISEASE SYNDROMES
DANIEL P. MOLDEN, GARY L. KLIPPLE, CAROL L. PEEBLES, ROBERT L. RUBIN,
ROBERT M. NAKAMURA, and ENG M. TAN
A distinctive type of speckled antinuclear antibody staining pattern was identified by indirect immunofluorescence on mouse kidney substrate in 4.8% of
5,976 specimens analyzed for antinuclear antibodies.
This pattern, termed variable large speckles (VLS),
consisted of 3-10 nuclear speckles ranging in size from
approximately 0.2-2.0~. The pattern could be differentiated from other indirect immunofluorescence patterns
related to specific antibodies. The predominant immunoglobulin isotype demonstrating the VLS pattern
was IgM in 27 of 28 sera examined and IgG in 1 serum.
VLS sera had substantial IgM antibodies to histone
demonstrated by enzyme immunoassay, and further
analysis of representative sera showed predominant
antibody activity to histone class 3 (H-3). Adsorption
with histone H-3 resulted in decrease or removal of
antibody producing the VLS pattern. Available informatiofi showed that most patients with LgM antibodies
of the VLS pattern had undifferentiated connective
tissue disease symptoms. They were characterized by a
heterogeneity of chronic symptoms including arthralgias, myalgias, inflammatory polyarthritis, myo-
Publication number 3680BCR from the Research Institute
of Scripps Clinic, La Jolla, California.
From the W. M. Keck Foundation, Autoimmune Disease
Center, Department of Basic and Clinical Research, Scripps Clinic
and Research Foundation, La Jolla, California.
Supported in part by NIH grants AM-32063, AM-34358,
and AI-10386.
Daniel P. Molden, MD: Clinical Fellow; Gary L. Klipple,
MI): Clinical Fellow; Carol L. Peebles, MS: Research Technician;
Robert L. Rubin, PhD: Assistant Member; Robert M. Nakamura,
MI): Member; Eng M. Tan, MD: Member.
Address reprint requests to Eng M. Tan, MD, Department
of Basic and Clinical Research, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037.
Submitted for publication February 11, 1985; accepted in
revised form June 14, 1985.
Arthritis and Rheumatism, Vol. 29, No. 1 (January 1986)
sitis, sicca symptoms, and pleurisy associated with elevation of the erythrocyte sedimentation rate. It remains
to be determined whether the IgM anti-histone H-3
profile of these patients is a transient or long-standing
serologic characteristic.
The indirect immunofluorescence technique is a
widely used method of screening for the presence of
antinuclear antibodies (ANA). The patterns of nuclear
fluorescence observed with this assay are indicative,
but not diagnostic, of a specific type of antibody (1).
There are many ANAs which display a variety of
speckled staining patterns; among these are antibodies
to Sm, RNP, SS-A (Ro), and SS-B (La). A speckled
pattern which can be distinguished from others is that
of the antibody to centromere/kinetochore (2). We
report here another distinctive type of speckled pattern which is different from other speckled patterns
and was first observed when mouse kidney sections
were used as the substrate for the indirect immunofluorescence test. It consists of 3-10 distinct, variably
shaped, 0.2-2.0~ nuclear speckles and will be referred
to as the variable large speckled (VLS) antinuclear
antibody pattern. Our studies provide information on
the frequency, the clinical significance, the substrate,
and the immune specificity of the VLS pattern.
PATIENTS AND METHODS
Patient sera. Of the 5,976 sera sent to this laboratory
for routine ANA testing, using mouse kidney sections as
substrate, 289 (4.8%) were observed to have multiple, variably sized fluorescent nuclear speckles. They were classified
as having a “variable speckled” ANA pattern. Titration data
were available on 210 of these 289 sera, and 81 of the 210
40
(38.6%) had titers of 1: 16 or greater. The remainder had
titers of 1:4 and 1 :8.
Since this was a retrospective study which focused
on sera that were found to be positive for the VLS pattern of
staining, clinical data on the patients were not readily
available. A questionnaire was designed and sent to physicians who had referred sera for analysis. The questionnaire
was sent only for those patients in whom titers were 1 : 16 or
greater. Sufficient information was returned on 38 of 81
patients, and these data formed the basis for determining the
possible correlation of symptoms or disease entities with the
serologic findings.
Determination of antinutlear antibodies by indirect
immurrofluorescence. An indirect immunofluorescence (IF)
technique for determining antinuclear antibodies was performed as described previously (1). Several tissues from
mice, iis well as tissues from rats, dogs, rabbits, and humans,
were used as substrates. All tissues were snap-frozen in dry
ice-acdone, and 4~ sections were prepared using a cryostat
microtome. Human peripheral blood lymphocytes were separated in a Ficoll-Hypaque density gradient (3) and either
applied directly to a glass slide coated with 1% bovine serum
albumin using a cytocentrifuge or cultured in RPMI 1640
with phytohemagglutinin and then cytocentrifuged onto
glass slides. WiL2 cells were cultured in 10% fetal bovine
serum in RPMI 1640 and also cytocentrifuged onto glass
slides. All of the above substrates were fixed briefly (1-2
minutes) in acetone at room temperature immediately before
testing. In most experiments, serum dilutions were 1:4,
1:16, 1:64, and 1:256.
A fluorescein isothiocyanate-conjugated goat antiserum to human Cohn fraction I1 (4) (fluorescein: protein
ratio 2.5) was used at a dilution of 1 :25 for ANA screening.
Immunoglobulin class specificity was determined with classspecific antisera obtained from Calbiochem-Behring (La
Jolla, CA) and used at 1 :20 dilution. Anti-IgM conjugate
(Tago, Burlingame, CA) at a 1 :100 dilution was also used for
some experiments. Evaluations were made at 6 2 5 ~magnification using a Leitz Ortholux I1 incident light microscope
with a 2OOW mercury light source, KP 490 excitor filter, and
K530 interference filter.
Immunodiffusion studies for precipitating antibodies.
The sera were tested for the presence of antibodies to Sm,
U1 RNP, SS-A (Ro), and SS-B (La) by a modification of the
Ouchterlony double-diffusion technique (5). Antibodies to
Sm and U1 RNP were detected using a saline extract of
rabbit thymus acetone powder (Pel-Freez Biologicals,
Rogers, AR) (5). Antibodies to SS-A (Ro) and SS-B (La)
were determined using an extract of WiLz cells prepared
from cultures maintained in the laboratory (6).
Selected sera were used as reference standards in the
immunodiffusion procedures. The Sm, U1 RNP, SS-B (La),
rheumatoid arthritis nuclear antigen (RANA), and SS-A (Ro)
prototype sera have been previously described and have
been used as standards itl this laboratory for several years
(7). Standards for anti-Sm, anti-U1 RNP, and anti-SS-B
(La), obtained from the Centers for Disease Control (Atlanta, GrA) were also used to corroborate specificity (8).
]Enzyme-linked immunosorbent assay (ELISA). Antibodies to native DNA (nDNA), single-stranded DNA
(ssDNA), total histones, and individual histones were as-
MOLDEN ET AL
sayed by techniques described previously (9,lO) using
peroxidase-conjugated antiimmunoglobulins (Tago).
Histones were isolated from calf thymus; their quality has
been previously shown (10). A serum dilution of 1: 100 was
used in the latter assays. Sera which gave negative IF-ANA
results on mouse kidney, as well as on Hep-2 and KB human
epithelial cell lines at a 1:20 dilution, were used as negative
controls for ELISA.
Adsorption of sera with histone H-3. Immulon I1
plates (Dynatech, Alexandria, VA) coated with purified H-3
at 1 pg/ml were used as immunoadsorbents. To determine
the appropriate dilution at which to perform the adsorptions,
sera were assayed by ELISA for H-3 antibodies over a range
of serum dilutions. A dilution which produced an optical
density just below 2.0 was used for adsorption studies. Each
diluted serum was added to 1 row of 6 wells and then
transferred through a series of 5 additional rows over a total
period of 16 hours. After adsorption, the sera from all wells
were pooled and concentrated approximately 100-fold in a
Minicon concentrator (Amicon, Lexington, MA). The final
dilution of each serum was monitored, with a small amount
of human '"I-IgG added prior to adsorption. Control for
nonspecific adsorption was carried out by transferring sera
through a parallel series of gelatin-coated wells.
Enzyme digestion and extraction experiments. Tissue
sections were fixed in acetone at room temperature. Fifty
microliters of either 100 p g h l DNase I or RNase A (Millipore, Bedford, MA) or 10 &ml of trypsin (Worthington,
Freehold, NJ) in various buffers was incubated on tissue
sections for 10 minutes (11). In previously reported studies,
it was shown that treatment with 1N HCl resulted in the
extraction of histones from nuclei, leaving behind a substrate
of histone-free DNA (1 l), and tissue sections treated in this
way were also used as substrates. Slides were then washed
in phosphate buffered saline (PBS), and IF-ANA was carried
out as described above.
Assay for cold agglutinins. Sera were separated from
the clot at room temperature and serially diluted in PBS to a
dilution of 1: 128, incubated for 1 hour at 4°C with human
group 0 erythrocytes, and observed for agglutination immediately upon removal from the cold (12).
Rheumatoid factor test. Latex agglutination titers for
rheumatoid factor were determined by a tube dilution
method (Calbiochem-Behring) (13).
RESULTS
Definition of VLS. The VLS immunofluorescence pattern was defined as 3-10 variably shaped,
distinct nuclear speckles ranging in size from 0.2-2.0p,
shown on mouse kidney substrate (Figure 1). This
pattern can be distinguished from the much finer
speckled patterns produced by Sm, RNP, and SS-B
antibodies, from the discrete speckles due to
centromere antibodies, and from the nucleolar pattern
of antibodies reactive only with nucleolar antigens
(Figure 1).
IgM ANTI-HISTONE H-3 ANTIBODY
41
Titer and immunoglobulin class. Fifty-one of the
81 sera studied (63%) had antibodies producing a VLS
pattern at titers of 1 :64 or greater. The remaining 30
sera (37%) had titers of 1: 16 or 1:32. Where sufficient
sera were available, further studies were performed on
those with antibody titers L 1:64, to determine the Ig
isotype and specificity of the antibody. Twenty-seven
of 28 sera studied had predominantly or exclusively
IgM-class VLS antibodies (Table 2). Only 1 serum (JS)
was found, by indirect immunofluorescence, to contain predominantly IgG VLS antibody. Five of the 27
sera with IgM antibody had IgG antibody at lower
titers, and there was only 1 serum with IgA antibody.
Antigen specificity. To study the chemical nature of the antigen responsible for the VLS pattern,
DNase, RNase, or trypsin digestion, or hydrochloric
acid extraction of the mouse kidney substrate was
carried out. The VLS pattern was removed by digestion of the substrate with DNase or trypsin and by HCI
extraction, but remained after digestion with RNase.
None of 81 sera analyzed by dodble immunodiffusion was found to contain antibodies to Srn, RNP, or
SS-B (La). Four of 81 sera contained anti-SS-A (Ro),
and 4 others had significant titers of anti-RANA. No
nucleolar or centromere antibodies were detected. No
Figure 1. Indirect immunofluoresence on mouse kidney sections as
substrate. a, Fine speckled antinuclear antibody pattern due to Sm,
RNP, or SS-B antibodies. This pattern is distinctly different from the
variable large speckled pattern (b), where 3-10 variably sized and
shaped distinct nuclear speckles are present (arrows). It is also
c1e:arly different from anticentromere (c) and antinucleolar (d)
staining.
Table 1. Presence or absence of variable large speckled (VLS)
staining pattern in tissues and cell cultures from several species
Species
Mouse
Substrate specificity. To examine the substrate
specificity of the antigen responsible for VLS, 10
randomly selected VLS sera with titers > 1: 16 were
used to stain a variety of cells and tissues. VLS
staining was found in all cells and tissues from several
animal species examined, with the exception of lymphocytes from thymus, spleen, lymph node, and peripheral blood (Table l). Lymphocytes from a variety
of tissue locations were all consistently negative for
VLS staining, despite the presence of VLS antigen in
splenic reticuloendothelial cells and thymic epithelial
cells. The antigen did not appear at any time in human
peripheral blood lymphocytes over a period of 5 days
following phytohemagglutination stimulation. VLS
staining was not found in WiL2 cells (a human B
lymphocyte tissue culture line) or in a human embryoriic lung fibroblast cell line.
Rat
Dog
Human
Rabbit
Cultured cells
VLS present
Kidney
Liver
Pancreas
Stomach
Testis
Heart
Brain stem
Bt-ain frontal lobe
Cerebellum
Thymus
(epithelial cells only)
Spleen
(reticuloendothelial
cells only)
Kidney
Kidney
Kidney
VLS not present
Thymus lymphocytes
Spleen lymphocytes
Lymphocytes
Phytohemagglutininstimulated lymphocvtes
Spleen
Lymph node
Human embyonic lung
cells
WiL2 cells (B lymphoWe)
MOLDEN ET AL
42
cold agglutinins were detected in any of 10 sera tested;
I
I
0
Anti-IgG
Anti-lgA
RA
PB
HC
BD
TD
RD
JD
DE
CG
EG
DOH
DaH
MJ
MN
MP
BR
TR
JS
1 :256
1:64
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
1:16
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
1:4
Neg.
Neg.
Neg.
Neg.
Neg.
sw
FC
ME
JM
LF
RG
JG
BH
1:256
1:256
1:256
1:256
1:256
1:256
1:256
1:256
1:256
1 :256
1:64
1:256
1 :256
1:64
1:256
1:256
Neg.
1:64
1 :256
1:256
1:256
1 :64
1:16
1:64
1:64
WJ
1:16
LL
1:256
* VLS
1:64
Neg.
1:256
Neg.
Neg.
1:16
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
= variable large speckled pattern; Neg. = negative.
I
I
0
I
0
I
*
I
I
0
1
!
0
I
I
Anti-IgM
1
I
Y
Patien1
IgG
I
I
IgG and IgM antibodies to nDNA, ssDNA, and
total histones were assessed by ELISA, and the results ;are shown in Figure 2. All the VLS sera had IgM
antihistone antibodies. IgM anti-ssDNA was also commonly elevated, but significant reactivity with DNA
was detected in only 2 samples. IgG antibody to these
antigens was generally within the normal range, except
for 2 sera with elevated IgG anti-ssDNA.
Figure 3 shows the results of ELISA for antibodies to individual histones. All sera were assayed
for IgM individual histone antibodies, except for 1
serum (JS), which was tested for IgG antibodies to
individual histone classes since this was the lone
serum with predominant IgG ANA. Eight of the 10
sera were found to contain antibodies which reacted
more strongly with histone H-3 than with other
histones. The other 2 sera (PB and ME) demonstrated
strong antibody activity to H-3 as well as activity to
the other histone classes. The titer of the antibody
producing the VLS pattern by indirect immuno-
Table 2. Immunoglobulin isotype and titer of VLS antibody*
I
IgM
4 of 2!2 sera tested contained rheumatoid factor.
-
8
-
nDNA -ssONA histone
nDNA
ssDNA histone
Figure 2. Anti-native DNA (nDNA), anti-single-stranded DNA
(ssDNA), and antihistone activities in randomly selected sera demonstrating the variable large speckled pattern. IgM and IgG antibodies were detected with immunoglobulin class-specific peroxidase
conjugates by enzyme-linked immunosorbent assay. Horizontal
lines mark the cut-off levels of normal serum binding.
1:4
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
fluorescence correlated fairly well with the magnitude
of the anti-H-3 activity determined by ELISA.
The results of adsorption of 5 VLS sera with
purified H-3 are shown in Table 3. Adsorption with
H-3 removed most of the anti-H-3 from 3 of the 5 sera
tested (BR, ME, and EC). In the other 2 sera, anti-H-3
activity was still detectable but at substantially lower
levels than in the unadsorbed or gelatin-adsorbed sera.
When the H-3-adsorbed sera were reconcentrated and
assayed by indirect immunofluorescence on mouse
kidney, the VLS pattern was either absent or, in
patient EC, present in decreased intensity. In contrast,
the VLS pattern was present in both the gelatinadsorbed and nonadsorbed controls for each of the 5
sera tested. These results suggested that the same
population of antibodies was responsible for the VLS
staining and the anti-H-3 activity.
43
IgM ANTI-HISTONE H-3 ANTIBODY
2.c
I
1 .[
r
c
.-
v)
c
al
n
-m
.-c
0
P
0
Ir
I
L
HI H2r Hzn H3 Ha
HI H I . H Z B H, H,
H.C.
R.S.
P.B.
VLS titer: 256
VLS titer: 64
VLS titer: 64
H,
%A
H2,
H3
H,
H,
H2. HZ8 "3 "4
HI Hz. Hzn H3 Ha
B.A.
M.E.
VLS titer: 256
VLS titer: 64
L.E.
H.C.
G.T.
F.M.
J.S.
VLS titer: 16
VLS titer: 64
VLS titer: 16
VLS titer: 16
VLS titer: 256
Figure 3. Individual histone profiles for 10 sera demonstrating the variable large speckled
(VLS) pattern. Sera were assayed by coating plastic wells with purified individual histones
and performing an enzyme-linked immunosorbent assay. Profiles shown were obtained
using anti-IgM-conjugated antiserum (except for the profile of serum JS, which was
obtained using anti-IgG-conjugated antiserum). Corresponding antinuclear antibody titers
of VLS antibodies, obtained by indirect immunofluorescence, are also shown.
Clinical data. Sufficient clinical information was
collected on 38 patients (Table 4). A variety of symptoms were present, including arthritis, sicca symptoms, pleurisy, myopathy, and esophageal dysfunction. The most common laboratory abnormality other
than the presence of ANA was an elevated erythrocyte
sedimentation rate, which was found in approximately
half the patients. Diagnoses in the 38 patients were
diverse (Table 9, with rheumatoid arthritis and undifferentiated connective tissue disease most common.
Many patients were not given a classifiable diagnosis.
None of the patients had systemic lupus erythematosus.
DISCUSSION
An autoantibody producing an indirect immunofluorescence pattern consisting of 3-10 variably
shaped, large nuclear speckles ranging in size from
0 . 2 - 2 . 0 ~has been identified on mouse kidney substrate. Careful observation permitted distinction of the
VLS pattern from patterns associated with antibodies
MOLDEN ET AL
44
Table 3. Effect of adsorption with histone H-3 on VLS staining*
Indirect immunofluorescence
ELISA anti-H-3
Serum, source and
treatment
Normal control 1
Normal control 2
Patient BR
Nonadsorbed
Gelatin-adsorbed
H-3-adsorbed
Patient PB
Nonadsorbed
Gelatin-adsorbed
H-3-adsorbed
Patient NE
Nonadsorbed
Gelatin-adsorbed
H-3-adsorbed
Patient ME
Nonadsorbed
Gelatin-adsorbed
H-3-adsorbed
Patient EC
Nonadsorbed
Gelatin-adsorbed
H-3-adsorbed
Serum
dilution
OD
1:125
1:125
0.310
1 :1,000
1:1,000
1:1,000
0.296
1.627
1.486
0.374
Serum
dilution
-
VLS
intensity
-
1:25
3+
-
-
1:24
0
1:20
1:44
1:40
2-3
0
1:125
]:I25
1:125
>2.000
>2.000
1:125
1:125
1:125
>2.000
>2.oM)
0.705
]:I25
1:125
1:125
1.135
1.056
0.254
1 :6
1:20
1:16
1:125
]:I25
1:125
1.251
1.276
0.285
1:36
1-2+
2+
1:68
0-1
0.491
2+
+
1:3
2+
1:13
2+
0
1:13
1:20
3+
3+
0
+
* Five sera were diluted and adsorbed in an H-3-coated microliter plate. Aliquots of the same sera
were also adsorbed on a gelatin-coated plate (gelatin-adsorbed control). All samples were then tested
by enzyme-linked immunosorbent assay (ELISA) for IgM anti-H-3 antibody activity. Each aliquot
was then concentrated and examined by indirect immunofluorescence. VLS = variable large speckled
pattern.
to Sm, RNP, nucleoli, SS-A (Ro), SS-B (La), and
centromere/kinetochore antigens. The patterns due to
Sm, RNP, and SS-B antibodies differed from VLS
antibodies in that they produced finer, more numerous, more diffuse nuclear speckles. Centromere/
kinetochore antibodies produced smaller, discrete
specklles.
The antigen responsible for the indirect im-
Clinical and laboratory features in 38 patients whose sera
demomtrated the variable large speckled pattern
munofluorescence VLS pattern was found to be
non-species-specific and present in all tissues except
lymphocytes. Thymic, splenic, lymph node, and peripheral blood lymphocytes, as well as WiLz cells (a
lymphoid cell line), were all poor substrates for the
antibody responsible for the VLS pattern, as were
mitogen-stimulated human peripheral blood lymphocytes.
The VLS pattern appears to be generally due to
Table 4.
Mean age (range)
Male/female, no. (%)
InRamrnatory peripheral arthritis, no. (%)
Seropositive
Seronegative
Sicca symptoms, no. (70)
Pleurisy, no. (%)
Inflammatory myopathy, no. (%)
Esophageal dysfunction, no. (%)
Elevated erythrocyte sedimentation rate, no. (%)
55.3 (34-74)
11/27(29/71)
12 (32)
5 (13)
7 (18)
9 (24)
5 (13)
3 (8)
3 (8)
18 (47)
Table 5. Diagnoses in 38 patients whose sera demonstrated the
variable large speckled pattern
Rheumatoid arthritis
Undifferentiated connective tissue disease
Idiopathic pericarditis
Multiple strokes with circulating anticoagulant
Idiopathic thrombocytopenic purpura
Mixed cryoglobulinemia
Chronic leukocytoclastic vasculitis
Idiopathic pulmonary fibrosis
No classified diagnosis
5
5
1
1
1
1
1
1
22
IgM ANTI-HISTONE H-3 ANTIBODY
IgM antihistone antibodies, having preferential specificily for histone H-3. This conclusion is supported by
the presence of IgM antibodies to total histones in all
sera that showed a VLS pattern and by the observation that removal of antibody to H-3 in representative
sera reduced or eliminated VLS staining. Although the
predominant reaction of most of the examined sera
was with histone H-3, other histones were also
antigenic, but these were not tested for capacity to
adsorb VLS staining. However, not all antihistone
antlibodies produce VLS staining: antihistone antibodies induced by procainamide usually give homogenous
staining, although VLS staining is occasionally observed with these sera (unpublished observations).
The inability of lymphocytes to display VLS staining is
perplexing since H-3 is a ubiquitous histone and an
integral chromatin component. The morphologic basis
for the speckled nuclear staining is also unclear, but it
may result from aggregation or condensation of chromatin as a consequence of multivalent binding of
pentameric IgM molecules.
Many of these sera also contained IgM antibodies to ssDNA, but this activity would not be likely to
produce nuclear staining. Many previously reported
studies have shown that anti-ssDNA per se does not
demonstrate nuclear staining, and ssDNA is not a
normal component of cells. Elevated anti-ssDNA is
commonly observed in many rheumatic diseases (7)
and is often associated with IgM antihistone antibodies
in patients treated with procainamide (14). However,
none of the patients in the current study was known to
be taking autoantibody-inducing drugs such as
procainamide or hydralazine. The significance of the
association between antihistone and anti-ssDNA is
presently unclear.
Fritzler et a1 (15) recently described 2 separate
autoantibodies, referred to as NSpI and NSpII, which
result in immunofluorescent nuclear staining of mouse
kidney nuclei with a pattern very similar to VLS. Sera
were obtained from patients with rheumatic disease
syrnptoms. However, in contrast to the variety of
locations in which VLS staining was seen, only the
peritubular cell nuclei in the kidney showed nuclear
staining in their study. No staining was found in the
renal tubular epithelial cells. Neither NSpI nor NSpII
antibodies were predominantly of the IgM class. Altholugh conditions of enzyme digestion and acid extraction experiments were slightly different from those
used in our work, NSpI antibodies, unlike VLS antibodies, were found to be resistant to DNase and
45
trypsin, and NSpII differed from VLS by its resistance
to DNase. Antigen specificity of NSpI and NSpII
antibodies was not determined.
Nomoto and Sakai (16) have reported the occurrence of IgM antibodies resulting in a speckled
ANA pattern which, on mouse kidney substrate, appears similar to the VLS pattern. The substrate specificity is also similar to that of the VLS antibody in that
antibodies were detected in all tissues except lymphocytes. However, these antibodies differ from VLS in
several aspects. First, they were detected by indirect
immunofluorescence at 4°C only, whereas VLS antibodies are detected at room temperature. Second,
they reacted with the RNase-resistant component of
extractable nuclear antigen, which the authors suggested might be Sm antigen; none of the VLS sera
reacted with Sm by immunodiffusion. Third, all of
Nomoto and Sakai’s patients had IgA nephropathy,
whereas the VLS patients did not have renal symptoms.
Patients whose sera contained the VLS antibody were found to have a heterogeneous group of
chronic problems. These included arthralgias,
myalgias, inflammatory polyarthritis, sicca symptoms,
pleurisy, myositis, and elevation of the erythrocyte
sedimentation rate. No single abnormality was present
in the majority of the patients. The most common
diagnoses were undifferentiated connective tissue disease and rheumatoid arthritis. The remaining patients
had a variety of clinical problems which did not
conform to distinct diagnostic classifications.
The clinical significance of our observations
might be to call attention to the fact that a certain
number of patients with symptoms which cannot be
classified into any category of connective tissue disease have IgM antibodies to histone class 3. They lack
or have low levels of IgG antibodies, and the unique
presence of IgM autoantibodies may escape detection
because polyspecific anti-Ig sera used as detecting
reagents usually have stronger reactivity with IgG and
weaker reactivity with IgM. An interesting question
which arises is whether these patients ,maintain this
IgM antibody profile as a basic characteristic of their
disease condition or whether this feature might represent an early stage of disease, before a switch from
IgM to IgG takes place. Although this question cannot
be answered without more extensive prospective
followup, we have seen a few patients who have
maintained their IgM anti-H-3 profile for at least 2
years.
46
1. Nakamura RM, Tan EM: Autoantibodies to nonhistone
nuclear antigens and their clinical significance. Hum
F’athol 14:392-400, 1983
2. Aloroi Y, Peebles C, Fritzler MJ, Steigenvald J, Tan
E:M: Autoantibody to centromere (kinetochore) in
scleroderma sera. Proc Natl Acad Sci USA 77:16271631, 1980
3. Biorjum A: Separation of leukocytes from blood and
bone marrow. Scand J Lab Invest (suppl) 21:77-89, 1968
4. Fromrnhagen L: The solubility and other physiochemical properties of human gamma globulin labeled with
fluorescein isothiocyanate. J Immunol95:442-445, 1965
5. Morthway JD, Tan EM: Differentiation of antinuclear
antibodies giving speckled staining patterns in immiunofluorescence. Clin Immunol Immunopathol 1:
140-154, 1972
6. Alspaugh MA, Tan EM: Antibodies to cellular antigens
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