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Effect of systemic lupus erythematosus antibodies against DNA on rna synthesis.

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45
EFFECT OF SYSTEMIC LUPUS
ERYTHEMATOSUS ANTIBODIES
AGAINST DNA ON RNA SYNTHESIS
OLGA V. MAKAROVA
The majority of tested systemic lupus erythematosus (SLE)sera inhibited RNA synthesis in vitro on the
stage of RNA chain elongation. Two sera were also active
in inhibiting the binding of the enzyme to the template. No
correlation has been found between the sera activity in
filter radioimmunoassay, their specificity to double- or
single-stranded DNA, and the degree of RNA synthesis
inhibit ion.
Sera of systemic lupus erythematosus (SLE) patients contain various antibodies that interact with
double-strand DNA, single-strand DNA, double-strand
RNA, DNA-RNA hybrids, single-strand RNA, and
Poly(rA) (1-5).
The effect of these antibodies on various manifestations of biological activity of nucleic acids, in particular the template activity of DNA, is insufficiently understood. A study of the literature has revealed only two
rather conflicting reports concerning the effect of SLE
antibodies on the template activity of DNA.
Whitaker and Starr (6) studied the effect of such
antibodies on DNA and RNA polymerase reactions in
vitro and concluded that there was no inhibition. The
From the Institute of General Genetics, USSR Academy of
Sciences, Moscow, USSR.
Olga V. Makarova, M.D.: Junior Research Worker of the
Laboratory of Molecular Genetics of Bacteria and Phages, Postgraduate in Biochemistry, Institute of General Genetics, Moscow,
USSR.
Submitted for publication April 6, 1977; accepted May 24,
1977.
Arthritis and Rheumatism, Vol. 21, No. 1 (January-February 1978)
authors believe that this result occurs because enzymes
have a higher affinity to DNA than d o antibodies. By
contrast, Poverenny and Prozorov (7) found that SLE
antibodies that react with native DNA inhibited the
template activity of T2 DNA whereas antibodies to
denatured DNA did not.
The present report is concerned with the effect of
SLE sera on RNA synthesis in vitro.
MATERIALS AND METHODS
Sera. Five SLE sera (PO, G O , KT, K , and BN) were a
gift from Dr. Ermakova of the Children’s Disease Clinic, First
Moscow Medical Institute, and five sera (B, FJ, MJ, OC, LN)
were kindly provided by Dr. N. Tala1 (Veterans Administration Hospital, San Francisco, California). Antisera against
human IgG and IgM were obtained from Hamaleya Institute
of Epidemiology and Microbiology.
R N A polymerase (fraction IV, DEAE cellulose chromatography) was isolated from E coli 3.0SO according to
Chamberlin and Berg (8). T4 D N A isolated according to
Goldberg (9) served as a template for R N A synthesis. Tritiumlabeled E coli D N A was isolated as described by Boyce and
Setlow (10). Unlabeled E coli D N A was isolated according to
the method of Marmur (1 1). Calf thymus D N A was a gift
from Dr. Slyusarenko (Institute of General Genetics, Moscow). D N A was denatured immediately before the experiment
by heating for 10 minutes at 100°C followed by rapid cooling
in an ice bath. The hyperchromic effect was 20 to 25%.
R N A Synthesis Assay. R N A synthesis was measured as
sH-UTP incorporation into acid-insoluble products. Each
sample (0.3-0.5ml) contained: p mol/ml, 10 tris-HCI (pH 7.9),
-10 MgCI2,, 5 MnCI,, 50 KCI, 10 2-mercaptoethanol, 0.1
EDTA, 80 gg/ml each of ATP, GTP, CTP, UTP, 0.6 p Ci
MAKAROVA
46
antibodies was carried out as described by Tala1 ef al. (15).
Sera at doses corresponding to 40-60% retention in the radioimmunoassay were mixed with various amounts of unlabeled
native or denatured competing D N A (from 0.008 to 1 pg). The
mixture volume was brought to 0.1 ml with tris-HCI buffer.
After incubation at 37°C for 30 minutes, 0.01 ml (0.05 pg) of
'H-DNA was added and the incubation continued for 30
minutes at 37°C and then for another 30 minutes at 0°C. The
mixture was passed through nitrocellulose filter and the radioactivity was measured as described above. The complex formation was inhibited by E coli and calf thymus DNA to the same
degree.
Assay of Ribonuclease Activity. Serum RNase content
was determined as the increase of acid-soluble R N A after
incubation with the sera. Yeast R N A (Schuchardt) was dissolved in 0.01 M tris-HCI, 0.15 M NaCl buffer (pH 7.2) at 2
mg/ml. To I ml of R N A 0.1 ml of heated serum ( 1 5 - l : l O ) was
added and the mixture was incubated for 2 hours at 37°C and
then transferred to an ice bath. After addition of HCIO, to 5%
concentration, the mixture was incubated at 0°C for 30 minutes, centrifuged at I 1,000 x g and the adsorption of supernatant at 260nm was measured. Control samples were treated
in the same way except that 0.1 ml of buffer was added instead
of the serum. The RNase activity is expressed as percent of
acid-soluble RNA.
'H-UTP (specific activity 3 Ci/mM), 1 pg T 4 DNA, 2.5-4
pg RNA polymerase.
Before the addition to R N A polymerase assay, the sera
diluted five- to tenfold were heated for 30 minutes a t 68°C.
Sera of healthy persons or patients suffering from other diseases served as the controls. The assay samples were incubated
for 20 minutes at 30°C; 400 pg albumin and 5% trichloroacetic
acid (TCA) solution were then added and the samples were
filtered through R U F S nitrocellulose filters (SYNPOR, Czechoslovakia) with a pore diameter of 2.5 p . The filters were
washed with cold 5% TCA, dried, and counted on a Nuclear
Chicago MARK-I scintillation spectrometer.
Immunological Methods. Complement fixation assay
was performed according to Wasserman and Levine (12) with
denatured (0.1 pg) or native (0.1-5 pg) D N A of T 4 phage or E
coli. The sera were used at 150-1: 100 or higher dilutions. Gel
precipitation was performed according to Hartmann and Toilliez (13).
Filter Radioimmunoassay. The assay was carried out as
described by Attias ef al. (14): sera diluted five-fold in 0.01 M
tris-HCI (pH 8.0), 0.15 M NaCl buffer were heated at 68°C for
30 minutes. Native E coli *H-DNA (0.05 pg in 0.01 ml of the
same buffer) was then mixed with 0.1 ml of different dilutions
of the sera. The mixture was incubated at 37°C for 30 minutes
and then at 0°C for another 30 minutes, diluted with the buffer
and passed through HUFS nitrocellulose filters (SYNPOR)
(0.4 p pore diameter). The filters were washed with 20 ml of the
buffer, dried, and radioactivity was measured. Radioactivity of
0.05 pg E coli 'H-DNA was 8,000-12,000 counts per min
(cpm). Sera dilutions corresponding to 50% retention of the
label were determined.
Inhibition of Complex Formation. The assay of inhibition of complex formation between native labeled D N A and
RESULTS
Preliminary experiments did n o t show any complement fixation or precipitation activity in four tested
SLE sera ( P O , BN, GO, a n d LN) ( d a t a n o t shown). For
Normal
10
I
I
1 : 1000
I
1 : 500
I
1: 250
Sera Dilution
I
1: 100
I
1 :40
Figure 1. Retention of double stranded 'H-DNA of E coli on cellulose nitratefilters by S L E sera OC and K .
Increasing concentrations of SLE sera (OC and K) were added to 0.05 pg of E coli a H - D N A (radioactivity
added = 8.OOo cpm). Experimental details are given in MATERlALS A N D METHODS.
SLE ANTIBODIES
47
90 -
80
'
ci
c
.-
-
70-
TI
c
60
0
50
-
40
-
a
c
.g
.-
0
c
-=
30
20
10
I
I
I
0.025
0.075
Competing DNA, pg
i
0.15
I
0.3
Figure 2. Retention of' E coli 'H-DNA by OC and K sera in presence of unlabeled double- or single-stranded
DNA . Increasing concentrations of unlabeled double-stranded (-)
or single-stranded (- - - -I competing
DNA werepreincubated with sera OC or K . then narioe E coli 'H-DNA was added (0.05 pg, 8,000 cpml. For
experimental details see
M A n u i A L s AND METHODS
this reason all the sera were tested in the radioimmunoassay (see MATERIALS
A N D METHODS).
Figure 1 shows retention of native E coli DNA as
a function of serum concentration for two sera (K and
OC). As can be seen from the figure, the retention is
proportional to the sera concentration. Pooled control
sera (1:40) fixed not more than 10%of labeled native E
coli DNA, whereas the retention by SLE sera at the
same dilution was up to 60%.
These experiments, however, do not exclude the
presence of anti-denatured DNA antibodies in the SLE
sera. To examine this possibility, the sera were tested in
the competition assay. Figure 2 shows that pre-incubation of OC serum with unlabeled denatured E coli
DNA resulted in a 15 to 50% decrease of 3H-DNA
retention whereas native E coli DNA inhibited the retention by 50 to 100%. It can be concluded, therefore, that
OC serum contains predominantly antibodies against
native DNA. In the case of K serum, however, native
and denatured competing DNA were equally effective.
To confirm the immunologic mechanism of
DNA retention by the sera, anti-IgG or anti-IgM was
added to the incubation mixture.
Table 1 shows that the serum against human IgG
at doses 240 and 100 pg protein inhibits filter retention
of E coli DNA by KT serum by 50 and 35%, respectively, whereas anti-IgM serum (100 pg) inhibits the
retention of E coli DNA by 12%. I n the control experiment, anti-IgG and anti-IgM produced no effect on T4
DNA retention by rabbit antiserum against T4 DNA.
The results of analysis of ten S L E sera are presented in Table 2. By their behavior in the radioimmunoassay, the sera may be divided into two groups:
1 ) those predominantly reacting with native DNA, and
2) those with equal reactivity toward native and denatured DNA.
I n the next series of experiments the effect of SLE
sera on the RNA polymerase reaction was studied.
As can be seen in Table 2 (columns 4,5,6), the
sera studied can be placed in the following order according to their activity in inhibiting RNA synthesis: LN >
OC > MJ > FJ. A comparison of these results with
those of the radioimmunoassay and the competition
assay reveals no apparent correlation for these properties of the sera. For example, the sera that show little or
no inhibition of RNA synthesis (KT, PO, GO, B) are,
MAKAROVA
48
Table 1. Effect o j Antisera Against H u m a n I g C and IgM on rhe
/ t r t l w i ~ ~ t i i 0/
~ t rA 7 SLE Serunr hYth E coli D N A *
AntiSerum
~~
~
IgG
"DNA
Filter
Retention of
Anti'H D N A . Inhibition.
IgMt
cpm
?I
~
240
SKE KT
E coli
100
50
25
-
Rabbit
anti-T4 D N A
240
-
T4
I 00
240
8.450
3.935
5.483
7.281
7.985
7 -473
527
532
575
52
35
14
6
I2
0
0
* S L E serum K T was incubated a t 37°C for 30 minutes with anti-IgG
o r anti-IgM in the total volume of0.l ml:0.01 ml o f 3 H - D N A ( 0 . 0 5 p g )
was then added and subsequent incubation was carried out a s deA N D METHODS.Antiserum against T4 D N A a n d
scribed in MATERIALS
T 4 3H D N A was obtained as described earlier (17). T h e dose of K T
serum used i n this experiment retained 65% E coli 'H-DNA o n t h e
filter while the dose of rabbit anti-T4 D N A serum retained 45Y of
T4 DNA.
t pg/sample.
nevertheless, highly reactive with respect to native as
well as denatured D N A . On the other hand, among the
four sera that markedly inhibit RNA synthesis there is
one (OC) reacting mainly with double-strand D N A
whereas the other three sera (LN, MJ, FJ) are equally
reactive with native and denatured E coli D N A .
These data suggest the presence in SLE sera of at
least two types of antibodies, one of which can inhibit
RNA synthesis and the other cannot.
The following experiments attempt to determine
the stage of RNA polymerase reaction that is inhibited
by SLE sera. Table 3 shows that four sera inhibit RNA
polymerase reaction irrespective of the time when they
were added to the incubation mixture, that is, before
incubation of D N A with the enzyme, after that, or after
incubation of D N A with the enzyme in the presence of
three nucleosidetriphosphates. In the case o f two SLE
sera (LN and B), the inhibition was two times lower
when the sera were added to the incubation mixture
after incubating D N A with RNA polymerase, that is,
after binding of the enzyme to the template. This could
be explained by the presence in these sera of antibodies
that atrect this binding as well as antibodies that inhibit
the elongation stage. This explanation, however, does
not account for the data shown in Table 3 where the
inhibition by these sera was not decreased under similar
conditions of DNA pre-incubation with the enzyme, but
in the presence of 3 nucleosidetriphosphates.
DISCUSSION
The results of this study show that most of the
SLE sera studied (6 of 10) inhibit RNA synthesis on T4
Table 2. The SpecificirI~oJ SLE Sera and Their Inhihition o/' R N A Synrhesis
R N A Synthesis Inhibition
Serum
LN
KT
PO
GO
B
Dilution
Retaining 501'
Native Labeled
E coli D N A
on Filters
1:1000
I :500
I :200
1:125
1:75
BN
K
1:55
1 :55
1:50
MJ
FJ
Normal
1:30
1:20
oc
Coefficient o f
Inhibition*
Inhibition.
4
Serum
Protein
per Sample.
mg
0.98- I .22
0.84-1.6
0.79-1.2
1.22-1.9
2.16-6.9
I . 15-1.5
0.59-1.24
2-3.2
0.94-1.27
0.82-1 .o
35
0
0
14
40
25
60
65
65
37
I00
770
400
840
640
I200
840
250
370
370
%' Inhibition
+ p g Protein
RNase
Contentt
0.35
0
0
0.0 17
0.06
0.02
0.07
0.26
0.17
0. I
35
I5
N Tf
5.5
9.8
9.3
20
36
16
24
10
* Coefficient of inhibition is expressed a s a ratio o f percent inhibition by competing native D N A to percent inhibition by t h e same a m o u n t of denatured competing D N A . T h e figures show t h e range of ratios for
different doses of competing D N A used in o n e experiment. T h e doses of competing D N A a r e given in
MATERIALS
A N D METHODS.
t RNase content is expressed in percent of acid-soluble R N A after treatment of 2 m g R N A in a volume
of I ml with 1 rng of serum protein.
f N T = not tested.
49
SLE ANTIBODIES
DNA as a template. We believe that this effect is not
related to the action of nucleases that are probably
present in the sera. Dr. V. S. Mikoyan of this laboratory
measured DNase activity of SLE and normal sera using
the technique described by Shimada and coworkers (16)
and found no difference between the two groups (data
not shown). The DNase content in the both groups of
sera was lower than 0.005 pg/ml.
The data on RNase activity are shown in Table 2.
As can be seen, sera LN, OC, FJ, and K have the highest
level of RNase activity. However, no correlation between the extent of RNA synthesis inhibition and the
RNase activity of the sera can be found. Sera MJ and
KT have similar RNase activity but are markedly different by their ability to inhibit transcription: MJ is an
active inhibitor (% inhibition per pg of protein = 0.17,
Table 2), whereas KT serum shows no inhibition even at
high concentrations. In the case of sera MJ and FJ, there
is also no correlation between the two parameters.
Addition of higher concentrations of normal
serum to the incubation mixture (thus leading to higher
RNase content) also does not affect the level of RNA
synthesis, which is further evidence against the correlation between inhibition and RNase activity.
I t is therefore concluded that inhibition of RNA
synthesis by SLE sera cannot be attributed solely to the
presence of RNase in the sera. This is also supported by
o u r earlier results (17) that show that antisera to T4 and
SPOl phage DNA are highly specific, that is, inhibit
RNA synthesis only on homologous DNA as a template. Addition of even a high excess of heterologous
antisera does not inhibit RNA synthesis.
I n some cases sera showing high inhibition also
had high RNase level. It is to be assumed that for these
sera the inhibition resulted from the action of both the
antibody and RNase.
The effect of four studied sera (K, FJ, MJ, and
OC) on RNA synthesis is caused by the interference of
antibodies with RNA chain elongation (Table 3 ) . Two
sera (LN and B ) also appear to inhibit the binding of
RNA polymerase to DNA. This is suggested by the fact
that pre-incubation of DNA with the enzyme lowers the
inhibition by antibodies. This phenomenon might result
from partial inaccessibility of antigenic sites in DNA
after binding of the enzyme. Conformational changes of
DNA may also contribute to the protective effect of the
enzyme. This question, however, requires more detailed
studies.
Our experiments did not reveal any correlation
between the three studied activities of SLE sera: 1 ) retention of double-stranded DNA on the filters, 2) specificity to double- or single-stranded DNA, and 3 ) inhibi-
5
(5t
z
p
MAKAROVA
50
tion of R N A synthesis (Table 2). It is noteworthy that
all the sera that inhibit R N A synthesis affect R N A chain
elongation and in this respect are similar t o D N A antibodies induced by immunization of animals.
Thus, it has been shown by Adler and coworkers
( I 8,19) and in this laboratory (17) that antibodies produced after immunization of animals with calf thymus,
E coli, and phage DNA that are known to react with
single-stranded D N A (1,2) inhibit R N A chain elongation and initiation.
The absence of correlation between the different
types of activity o f sera remains unexplained. It may be
supposed that antibodies of different sera vary by their
affinity to DNA. The conclusions drawn require some
comments since in these experiments we used D N A of
T4 phage as a template. This D N A has been chosen
because it provides a convenient model system for
studying antibody effect on transcription (17). It is possible that somewhat different results would be obtained
if we used mammalian D N A as a template. Further
studies are needed to solve this problem.
ACKNOWLEDGMENTS
I am deeply indebted to Professor D. M. Goldfarb and
Dr. L. A. Zamchuk for their valuable advice and to Dr. V . S.
Mikoyan for the assistance in preparing the manuscript for
publication. Thanks are also due to Dr. N. Talal and T. M.
Ermakova, M.D. who kindly provided the sera.
REFERENCES
1. Stollar BD: Nucleic acid antigens, The Antigens, Vol I .
Edited by M Sela. New York, Academic Press, 1973, pp
1-75
2. Stollar BD: The specificity and applications of antibodies
to helical nucleic acids. C R C Crit Rev Biochem 3:45-69,
I975
3. Talal N: Antibodies to nucleic acids in human and murin
lupus. J Rheumatol 2:130-138, 1975
4. Talal N, Gallo RC: Antibodies to a DNA: RNA hybrid in
systemic lupus erythematosus measured by a cellulose
ester filter radioimmunoassay. Nature New Biol
240:240-242, I972
5. Pillarisetty RJ, Talal N: Clinical studies of antibodies
binding polyriboadenylic acid in systemic lupus erythematosus. Arthritis Rheum 19:705-710, 1976
6. Whitaker J N , Starr JL: In vitro effect of antibodies to
DNA on the template activity of DNA. J Clin Invest
47: 1496-1 5 10, 1968
7. Poverenny AM. Prozorov AA: Effect of antibodies from
blood serum of patients with lupus erythematosus upon
specific functions of DNA. Voprosy Med Khimii (USSR)
1211 17-1 19, 1966
8. Chamberlin M, Berg P: Deoxyribonucelic acid-directed
synthesis of RNA by an enzyme from E coli. Proc Nat
Acad Sci USA 48:81-94, 1962
9. Goldberg E: The amount of DNA between genetic markers in phage T4. Proc Nat Acad Sci USA 56:1457-1463,
1966
10. Boyce RC, Setlow RB: A simple method of increasing the
incorporation of thymidine into the DNA of E coli. Biochem Biophys Acta 61:618-620, 1962
1 I . Marmur J: A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208-218,
1961
12. Wasserman E, Levine L: Quantitative micro-complement
fixation and its use in the study of antigenic structure by
specific antigen-antibody inhibition. J Immunol 87:290295, 1961
13. Hartmann L, Toilliez M: Micro-mkthode d’ttude en gelose de la reaction antigtne-anticorps (Variante du proc t d t d’ouchterlony). Rev Franc Etude Clin et Biol
21197-199, 1957
14. Attias MR, Sylvester RA. Talal N: Filter radioimmunoassay for antibodies to reovirus RNA in systemic
lupus erythematosus. Arthritis Rheum 16:7 19-725, 1973
15. Talal N. Steinberg AD, Daley G: Inhibition of antibodies
binding polyinosinic. polycytidylic acid in human and
mouse lupus sera by viral and synthetic ribonucleic acids.
J Clin Invest 50:1248-1252, 1971
16. Shimada K, Nishimoto T, Takagi Y: A method for the
detection of strand breaks in DNA samples. J Biochem
75:611-617, 1974
17. Makarova OV, Polonsky YuS, Zamchuk LA, Zograf
YuN: Influence of proteins bound with single-stranded
D N A on the synthesis of RNA and poly(A). I. Antibodies
to DNA. Molek Biologiya (USSR) 9361-870, 1975
18. Adler V, Poverenny A, Podgorodnichenko V, Shapot V:
Inhibition of in vitro transcription by antibodies to DNA.
lmmunochemistry 10:555-558, 1973
19. Adler VV, Poverenny AM, Podgorodnichenko VD, Shapot VS: Study of the process of transcription using antibodies to DNA. Molek Biolgiya (USSR) 7:203-21 I , 1973
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