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The role of histones as nuclear autoantigens in drug-related lupus erythematosus.

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1064
THE ROLE OF HISTONES AS
NUCLEAR AUTOANTIGENS IN
DRUG-RELATED LUPUS ERYTHEMATOSUS
ENG M. TAN and JOSEPH P. PORTANOVA
Patients with drug-related lupus erythematosus
produce antibodies to nuclear histones which can be detected by a three-step indirect immunofluorescent technique. Procainamide-related antinuclear antibodies
were detected by this technique, but hydralazine-related
antinuclear antibodies were not. Certain evidence suggests that antibodies induced by the two drugs are reactive with Merent subclasses of histones. Hydralazine
was shown to interact with a soluble DNA-histone complex, and the resulting interaction rendered the histone
moiety resistant to trypsin digestion. This mechanism
may help to maintain DNA-histone complexes in a potentially immunogenic form and result in the production
of autoantibodies.
ities, including anti-DNA and antibodies to nonhistone
antigens, whereas in drug LE antibodies to native DNA
and nonhistone nuclear antigens were not detectable
(1). These two observations, i.e. presence of antibodies
to histones and absence of antibodies to other nuclear
antigens, have proved to be useful laboratory parameters in establishing the diagnosis of drug-induced lupus
and in distinguishing it from SLE.
In this report we will review some of the previous
observations and report the new finding of differences
in the serologic profiles of lupus induced by procainamide and hydralazine.
Antibodies to histones have been found in a
number of systemic rheumatic diseases including the
idiopathic form of systemic lupus erythematosus (SLE),
drug-induced lupus (drug LE), and rheumatoid arthritis
(1-4). In a previous study an unusual feature in drug-induced lupus was that antibodies to histones were present in almost all patients with drug LE (1). In contrast,
in the naturally occurring idiopathic SLE, antibodies to
histones were observed in approximately 25% of such
patients. A similar frequency appears to be present in
patients with rheumatoid arthritis (3). In addition, another distinguishing feature between the two types of
lupus was that SLE was characterized by the presence
of antinuclear antibodies (ANA) of multiple specific-
The materials and methods used in the studies reported here have been published in detail previously (5).
Briefly, a three-step immunofluorescent method was used for
the detection of antibodies to histones.
Mouse kidney cryostat sections fixed in acetone but
otherwise untreated were reacted with serum in the indirect
immunofluorescent method. The fluorescein conjugate consisted of antiserum to human IgG labeled with fluorescein
isothiocyanate. Serum containing antibodies to histones demonstrate a homogeneous nuclear staining pattern.
The second step consisted of a mouse kidney section
which was extracted with 0.1N HCl for 30 minutes at room
temperature. This treatment resulted in the extraction of histones from the nuclei in the tissue section. The HC1-extracted
section was then reacted with an aliquot of the same serum
sample. Because histones are absent from this acid-extracted
tissue section, the serum now becomes negative for ANA if
only antibodies to histones are present.
The third step consists of another acid-extracted
mouse kidney section to which purified histones have been
added. The added histones complex with the DNA present in
the nuclei, and this substrate is now used as a histone-reconstituted tissue section for reaction with another aliquot of
the same serum. the serum becomes positive again for ANA.
Thus, in the three-step method, a serum containing
antibodies to histones would be positive for ANA on the first
From the Division of Rheumatic Diseases, Department of
Medicine, University of Colorado Health Sciences Center, Denver,
Colorado 80262.
Supported by NIH Grant #AM20705 and a Clinical Research Center Grant from the Arthritis Foundation.
Address reprint requests to Eng M. Tan, MD, Division of
Rheumatic Diseases, University of Colorado Health Sciences Center,
Denver, Colorado 80262.
Arthritis and Rheumatism, Vol. 24, No. 8 (August 1981)
MATERIALS AND METHODS
ROLE OF HISTONES
1065
section, negative for ANA on the acid-extracted section, and
become positive again on the histone-reconstituted section.
The sera examined in this study were taken from patients who had a lupus-like clinical illness which could be directly related to ingestion of hydralazine or procainamide.
RESULTS
Figure 1 shows the detection of anti-histone antibodies by the three-step immunofluorescent method. A
consistent observation was that with the histone-reconstituted section, the pattern of ANA is frequently
nodular and not homogeneous. In many sera, the titer
of the ANA on histone-reconstituted sections is equal to
the titer on the untreated section. In some sera, the titer
on histone-reconstituted sections could be a small
amount lower.
In a previously reported study, the observations
on 23 patients with drug-induced LE were compared
with those on 20 patients with SLE. The results are depicted in Figure 2 for the three-step immunofluorescent
method. It is seen that all patients with drug LE became
negative for ANA on acid-extracted sections and with
the exception of one patient, the other 22 patients became positive for ANA again on histone-reconstituted
sections. The findings with SLE were strikingly different. Many sera became negative for ANA on acid-extracted sections, but there were several where the ANA
remained at the same titer or were only reduced in titer.
Of further interest was the finding that although a few
of these sera became positive for ANA on the histonereconstituted sections, the majority remained negative.
It could be demonstrated that the difference in
the findings between drug LE and SLE was related to
the fact that the sera of SLE patients contained antibodies of multiple specificities, whereas drug LE sera
contained mainly antibodies to histones (Table 1). In
addition to antibodies to histones, SLE sera contained
antibodies to Sm and nRNP antigens as well as to
double-stranded and single-stranded DNA. On the
other hand, 3 of 23 patients with drug LE had antibodies to single-stranded DNA, but antibodies for other
nuclear antigens except histones were not detected.
2 was exThe diversity ofpatterns Seen in ~i~~~~
plained on the following basis. Extraction of tissue sections with acid resulted in the removal of all or most
histones and several nonhistone protein antigens. HOWever, double-stranded DNA remained in the nuclei of
antibody to doublethese sections. sera
stranded DNA would remain positive on acid-extracted
sections, which was observed with several SLE sera. On
histone-reconstituted sections, the antigens present in
A
B
C
Figure 1. A three-step indirect immunofluorescent test for the detection of antibodies to nuclear histones. The substrate consisted of sections of mouse kidney. In A, sections were fixed in acetone and reacted with the serum from a patient with drug-related LE containing
to histones. A homogeneous pattern of nuclear staining
was observed. In the second step (B), a companion tissue section was
extracted with 0.1 N HCl to remove nuclear histones. The ANA test
of the same serum became negative. A third tissue section (C) was extracted with HCI to remove histones, and subsequently purified histones were added to the acid-extracted tissue section. Purified histones
complex with the DNA remaining on the acid-extracted tissue settion, and the reconstituted “section” shows positive ANA again with
the same serum.
TAN AND PORTANOVA
1066
-
1024-
0
I
IDIOPATHIC SLE
DRUG LE
512256-
.-c
128-
t
U
z
e
64-
\
c
3216-
...
&*A
AAb
Contra'
Acid
Extracted
Histone
Reconstitutet
Control
Acid
Extracted
I
I
.A
8 A.
A
Histone
Reconstituted
Figure 2. The three-step indirect immunofluorescent technique was used to analyze sera of patients with
drug-related LE and idiopathic SLE. Striking differences were observed. In drug LE, all sera became
negative for ANA on acid-extracted tissue sections, and except for one serum, they were all positive again
for ANA on histone-reconstituted sections. In contrast, some SLE sera remained positive for ANA on
acid-extracted sections and the majority did not regain positive ANA on histone-reconstituted sections.
The reasons for these differences are explained in the text.
nuclei now consisted of DNA and histones but were still
devoid of nonhistone antigens such as Sm and nRNP.
Therefore, sera containing antibodies to Sm or nRNP
would not become positive for ANA on the histone-reconstituted sections.
This method could also lend itself to the detection of antibodies to specific subclasses of histones. In
the study shown in Table 2, it was observed that the majority of drug LE patients had antibodies directed
against the H2A-H2B histone complex. There were 3 of
22 sera with antibodies to H3-H4 complex and 2 of 22
sera with antibodies to H1. Only 5 of the SLE sera
could be analyzed for antibodies to histone subclasses in
this fashion since the other 2 contained antibodies with
titers too low for this analysis. Of the 5 SLE sera, the
distribution of antibodies to histone subclasses appeared
to be more heterogeneous than that found in the sera of
the drug LE patients.
These observations were made with sera mainly
from patients with lupus syndrome induced by procainamide. In our current studies, we have examined
the reaction of hydralazine-induced LE sera. These new
findings are shown in Tables 3 and 4. In Table 3, 10 patients with pronestyl LE were studied for anti-histone
antibodies. It is seen that all sera became negative at a
titer of <4 for ANA on acid-extracted sections. With the
Table 1. Antibody profiles of drug-related LE and idiopathic SLE
Idiopathic SLE
Drug-related LE
Antibodies to
Total
tested
Sm
nRNP
Histones
dsDNA
ssDNA
20
23
9
0
3
0
7
22
7
5
0
3
ROLE OF HISTONES
1067
Table 4. Hydralazine-induced IgG antinuclear antibodies
Table 2. Reactivity with histone subclasses
ANA titer on mouse kidney
Number of sera reactive with
Idiopathic SLE
Drug-relatedLE
Total
histones
HI
H2A-H2B
H3-H4
5
22
2
2
3
21
I
3
exception of two sera, the other eight became ANA positive again on histone-reconstituted sections.
In contrast, the reaction of hydralazine-induced
ANA was quite different. First, the titers of ANA on acetone-fixed sections were generally lower than the ANA
titers of pronestyl-induced ANA. The hydralazine sera
all became negative for ANA on acid-extracted sections,
but the interesting finding was that they all remained
negative on histone-reconstituted sections. On untreated
tissue sections (i.e. acetone-fixed only), the ANA patterns of hydralazine sera were all homogeneous and
looked identical to that of pronestyl-induced AN A.
Likewise, the hydralazine sera were negative for ANA
on acid-extracted sections and, in these two characteristics, were similar to the pronestyl-induced ANA and
different from SLE sera. The negative test for ANA on
histone-reconstituted sections therefore appeared to
contrast pronestyl-induced LE from hydralazine-induced LE. In preliminary studies, we have evidence that
hydralazine-induced ANA contains antibodies to histones but is reactive with histones of Werent subclasses
from that of pronestyl-induced ANA. The three-step
immunofluorescent method appears to be detecting histone specificities associated with the H2A-H2B complex
and not other histone subclasses.
Some previous studies have been directed at
elucidating the possible mechanism relating to induction of anti-histone antibodies. It has been difficult to
analyze for precipitating antibodies to nuclear histones
Table 3. Pronestyl-induced IgG antinuclear antibodies
ANA titer on mouse kidney
Patient
-
Ta
Wh
Er
Ga
Gu
Ho
Mo
No
Pr
Om
Acetone
fixed
O.lNHC1
extracted
Histone
reconstituted
4096
4096
4096
2048
2048
2048
1024
1024
512
32
<4
t 4
<4
t 4
t4
<4
<4
<4
<4
t4
512
2048
512
1024
256
2048
256
<4
64
<4
Patient
Ra
we
Bo
Ma
Ba
Wh
Jo
Mi
LY
K1
Acetone
fixed
0.1N HC1
extracted
Histone
reconstituted
2048
512
256
256
I28
128
128
128
I28
4
<4
<4
<4
<4
t4
<4
<4
<4
t4
t4
t4
t4
<4
<4
t4
<4
<4
<4
t4
t4
by immunodiffusion methods because of nonimmunologic reactions between serum proteins and free histones. However, certain forms of histones can be isolated from nuclei in a soluble nucleoprotein (sNP)
complex, which has been shown to precipitate with antibodies in the sera of patients with SLE and hydralazineinduced LE (6). It was shown that the sNP antigen consists of DNA-histone complexes. The antigenicity of
sNP can be abolished by treatment with either DNAse
alone or trypsin alone. In experiments previously reported (7), sNP antigen was isolated from calf thymus
nuclei and increasing amounts of hydralazine were
added to a standard amount of sNP. The findings are
shown in Figure 3. There was a physicuchemical interaction between sNP and hydralazine in that the viscosity of this mixture increased with increasing concentrations of hydralazine. Experiments were also performed
with purified DNA and purified histones as illustrated
in Figure 3. There was some slight increase in viscosity
when hydralazine was added to purified DNA but no
increase in viscosity when hydralazine was added to purified (free) histones.
It was of interest to study some of the immunochemical characteristics of the sNP-hydralazine complex. As mentioned previously, the sNP antigen by itself
was labile to treatment with either DNAse or trypsin.
On the other hand, when sNP-hydralazine complex was
subjected to the same enzyme treatments, DNAse continued to destroy precipitating activity, but trypsin did
not. Therefore, the complexing of hydralazine with sNP
in some way protected the histone moiety of the sNP
complex from digestion with trypsin (Figure 4). It was
postulated from these studies that hydralazine interaction with nucleoprotein complexes in vivo might protect nucleoprotein from breakdown by proteolytic enzymes and thus create or maintain a potentially immunogenic autoantigen.
TAN AND PORTANOVA
1068
3.0
2.6
0
Is=
.s 2.2
v)
0
u
.-w
v)
aa
.P
w
-maa
1.8
a
1.4
Histones
._...
.......
......
1.oJ
2 4 6 8 1 0
Final Concentration Hydralazine
A
This finer differentiation of antibodies to histone
subclasses and their corresponding relationships with
procainamide or hydralazine bring up intriguing questions concerning the mechanisms responsible for their
induction. Many investigators at this conference have
discussed and analyzed the chemical interactions between procainamide or hydralazine (and their metabolites) and nuclear macromolecules. If these interactions
should occur in vivo in patients ingesting these drugs
and this interaction stimulates an immunologic response, our studies indicate that the nuclear macromolecules of interest should be histones. This is based on
the finding that most patients with drug-related LE
have antibodies to nuclear histones.
On the other hand, it is possible that these drugs
react in vivo with other nuclear macromolecules such as
DNA and nonhistone proteins, and such interactions
may promote subsequent interaction with histones. If
drug-DNA interaction occurs, it does not appear to be
very immunogenic since antibodies to double- or even
single-stranded DNA are infrequently detected. Antibodies to certain nonhistone nuclear proteins, however,
have been detected in patients treated with pro-
[mg/mll
Figure 3. Hydralazine was added to soluble nuclear protein (sNP)
and to calf thymus DNA and calf thymus histones. The relative viscosity of the mixtures was measured in an Ostwald viscosimeter and
plotted relative to viscosity without addition of hydralazine. The viscosity of sNP-hydralazine mixtures increased significantly with increasing concentration of hydralazine. There was a slight increase in
the viscosity of DNA-hydralazine mixtures but no increase in viscosity with the histone-hydralazine mixtures.
DISCUSSION
Recent studies have confirmed the usefulness of
the three-step immunofluorescent technique for the detection of antibodies to histones. In almost all patients
with procainamide-induced lupus, this antibody is present to the exclusion of antibodies to native:DNA and
nonhistone proteins. The recent studies comparing procainamide-induced ANA with hydralazine-induced
ANA have uncovered some interesting differences.
Hydralazine-induced ANA is not detected with the immunofluorescent technique, but other evidence points to
the conclusion that hydrdazine-induced ANA are also
antibodies to histones. However, these antibodies are
not directed against the H2A-HZB complex, which is
the antigenic determinant for the procainamide-induced
ANA.
Figure 4. The precipitating activity of sNP is destroyed by either
DNAse or trypsin treatments as demonstrated in the immunodiffusion study shown in the upper frame. The center well contained
seTum from a patient with SLE who had precipitating antibody to
sNP. The observations with sNP-hydralazine mixtures are shown in
the lower frame. The sNP antigen in the hydralazine mixture continued to be labile to DNAse treatment but was now resistant to trypsin.
ROLE OF HISTONES
cainamide (8). This has some features of a secondary
immune response since antibodies are detected very
shortly after the drug is started, but the immune response is short-lived and antibodies are not detectable in patients continued on the medication.
1069
4.
5.
REFERENCES
Fritzler MJ, Tan EM: Antibodies to histones in drug-induced and idiopathic lupus erythematosus. J Clin Invest
62560-567, 1978
Stollar BD: Reactions of systemic lupus erythematosus sera
with histone fractions and histone-DNA complexes. Arthritis Rheum 14:485492, 1971
Aitcheson C, Peebles C, Joslin F, Tan EM: Characteristics
6.
7.
8.
of antinuclear antibodies in rheumatoid arthritis. Arthritis
Rheum 23528-538, 1980
Agnello V, Arbetter A, Ibanez de Kasep R, Powell R, Tan
EM, Joslin F: Evidence for a subset of rheumatoid factors
that cross-react with DNA-histone and have a distinct
cross-idiotype. J Exp Med 151:1514-1527, 1980
Tan EM, Robinson J, Robitaille P: Studies on antibodies to
histones by immunofluorescence. Scand J Immunol 5:811818, 1976
Tan EM: An immunologic precipitin system between soluble nucleoprotein and serum antibody in systemic lupus
erythematosus. J Clin Invest 46:735-742, 1967
Tan EM: Drug-induced autoimmune diseases. Fed Proc
33:1894-1897, 1974
Winfield JB, Koffler D, Kunkel HG: Development of antibodies to ribonucleoprotein following short-term therapy
with procainamide. Arthritis Rheum 18531-534, 1975
DISCUSSION
Dr. Shulman: Of the 20 patients with SLE, 6 had glomerulonephritis and the others did not. Were the 3 in
the SLE category in the nonrenal group? Did they
have an illness that might resemble drug-related
lupus?
Dr. Tan: No, they didn’t seem to. The incidence of antiDNA antibodies in that group of 20 patients was not
inconsistent with that of a larger group of patients
with systemic lupus erythematosus. We had about 8
or 9 patients with anti-Sm antibody. In general, there
was nothing in the idiopathic lupus that we could ascribe to any particular antibody specificity.
Dr. Hess: How do you explain the two pronestyl patients who reacted similarly to the hydralazine patients?
Dr. Tan: Not all procainamides are highly reactive with
2A-2B complex. Some are still reactive with H3-H4
complex, and we have 1 or 2 patients who are reactive
with H1 complex.
Dr. Parker: Is there any indication that the pattern
changes with time during the course of the illness?
Dr. Tan: We haven’t had a chance to follow these patients. Many have been referred only for a one-time
ANA analysis.
Dr. Alarcon-Segovia: It has been said that the LE cells
result from interaction of antibodies to nucleoprotein
with this material in disrupting cells. Does this antibody form LE cells?
Dr. Tan: I think that is true. We performed the indirect
LE cell test first described by Lachmann in which
only the serum is needed. From these studies we concluded that the LE cell phenomenon is related to
antibody to nucleoprotein. If antibodies to nucleoprotein are removed, the LE cell phenomenon is
negative.
Dr. Stollar: Did either or both of those react with isolated nucleosomes? The hydralazine-induced antibodies reacted with chromatin and with isolated histone. They must be able to react with some form of
complexed histone unless there are free histones.
Dr. Tan: It is clear that native tissue sections are positive
for ANA with hydralazine sera. Whatever 2A determinant they are reacting with is available on the
tissue section. In our reconstituted sections, the 2A2B complex reassociates with the DNA in a clumpy
fashion, and the 2A determinant may be hidden so
that it is not available for reaction with hydralazine
sera.
Dr. Stollar: Have you tried salt-extracted histones that
have never seen acid?
Dr. Tan: Yes, but it made no difference.
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