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The response of cultured lymphocytes from patients with systemic lupus erythematosus to DNA.

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The Response of Cultured Lymphocytes from Patients
with Systemic Lupus Erythematosus to DNA
By AIUA PATHUCCO,
NAOMI F. KOTHFIELD AND KURT HIRSCHHORN
that the immunologic responses of an animal are effected by
he activity of fixed and circulating cells,
which, in aggregate, make up the lymihoid tissue. The small lymphocytes, which
ire the predominant cells in this tissue,
lave been shown to have many potential
'unctions: They recirculate in the organism,
hey have a long life span, and, when
ippropriately stimulated, they develop into
arge pyroninophilic cells and begin to
clivide.'g2
During the past few years it has been
shown that human peripheral blood lymphocytes can respond to a variety of stimuli
in cell culture by enlarging and dividing.
These cells respond to specific antigens
to which the donor is sensitized, as well as
to certain nonspecific stimulants such as
phytohemagglutinin (PHA). It has been
suggested that this in vitro system may be
representative of the donor's immune
status.
The significance of immunologic reactions in the pathogenesis of systemic lupus
erythematosus (SLE) is obscure. Different
antibodies to nuclei, deoxyribonucleic acid
(DNA), histones, and nucleoproteins found
in this disease have been investigated, but
T IS KNOWN
their role in the clinical manifestations of
the disease has not been established."-"
The purpose of the present study was to
examine the in vitro response of peripheral
lymphocytes from patients with SLE when
these cells were cultured in the presence
of DNA. The results suggest that these
lymphocytes, unlike those of normal subjects, do respond to stimulation with DNA.
MATERIALS
AND METHODS
The cells were cultured by the method described
in detail elsewhere.7 Forty ml. of heparinized blood
was allowed to sediment at 37 C. The supernatant plasma was removed and left on glass at
37 C. for from one to two hours to allow
attachment of polymorphonuclear leukocytes and
monocytes to the glass. The supernatant was
carefully removed. This contained between 60 and
95 per cent small mononuclear cells. The cells
were washed three times and then suspended in
minimal essential Eagle's medium modified for
suspension culture* with 20 per cent fetal calf
serum, penicillin, streptomycin, and 1-glutamine.
The final cell concentration was 750,000 mononuclear cells per ml. Four ml. cultures were set
up in replicate tubes at 37 C., and 0.1 ml. of
PHA-Mf was added to one tube from each blood
obtained. Two control tubes were set up for each
blood which contained no additive. Duplicate tubes
containing 1.0 gamma/ml. of highly polymerized
calf thymus DNA$ were set up for each blood
Professor of Medicine, New York Uniuersit3
School of Medicine, New York, N . Y. KURI
HIRSCHHORN,
M.D.: Associate Professor of Medi.
cine, New York University School of Medicine
Career Scientist, New York City Health Researd
Council, 1-41 6, present address: Mt. Sinai Schoo
of Medicine, New York, N. Y.
OMEM-Spinner, Grand Island Biological Corn
pany, Grand Island. N. Y.
Difco Laboratories, Detroit, Mich.
4Mann Research Laboratories, New York, N. Y
From the Department of Medicine and the
Rheumatic Diseases Study Group, New York University School of Medicine, New York, N. Y.
This study w a s supported by grants from the
USPHS (HD-00542 and AI-068531, T h e New
York City Health Research Council Grant U-1030,
The American Heart Association, and The Lupus
Foundation, N e w York.
AIDA PATRUCCO,
M.D.: Fellow, Michael I . Schaffer Foundation. Present address: Hospital Privado,
Cordoba, Argentina. NAOMIF. ROTHFIELD,M.D.:
Clinical Scholar, Arthritis Foundation; Assistant
+
32
A N D RHEUMATISM,
VOL. 10, No. 1 (FEBRUARY,
1967:
ARTHRITIS
33
RESPONSE OF CULTURED LYMPHOCYTES
obtained. DNA was denatured by heating a t 100 C.
for ten minutes and then rapidly cooling. The
denatured DNA was added to culture tubes with
the final concentration of 1.0 gamma/ml. Cultures
containing PHA were harvested at three days, and
control and DNA tubes a t five days. After harvesting, the cells were washed in 1 per cent
sodium citrate, fixed in 3:l methanol: glacial
acetic acid, and stained with 0.5 per cent acetic
orcein. Slides were examined using phase contrast
illumination. One thousand cells from each culture
tube were classified as large, small, or in mitosis.
‘The degree of response was defined as the percentage of large cells plus cells in mitosis in the
experimental cultures less the percentage found
in the cultures without additive. A positive response was defined as an increase of 5 per cent or
more in the experimental tubes.8
In each instance, the blood was drawn, numbered, and clinical data obtained by one investigator. Cultures were set up and analyzed by
a second investigator without knowledge of the
diagnosis.
The fluorescent antinuclear antibody test was
carried ont as previously described using mouse
liver as a source of nuclei and rabbit antihuman
gamnia globulin conjugated to fluroescein isothiocyanate.9
Twelve patients with SLE were studied. All
patients had multiple system disease compatible
with SLE and positive LE cell tests at some time
during the course of the disease. Ten healthy
normal individuals and eleven patients with other
diseases were also studied. All patients were studied
either while they were patients at Bellevue Hospital or as outpatients in the New York University
Arthritis C h i c of Bellevue Hospital.
Table 1.-Response
to Phytohemagglutinin
% Increase of Large
Cells and Mitoses
NO.
SLE
Rheumatoid
Arthritis
Other Diseases
Normal
Studied
Range
Mean
14
60.1-80.2
74.6
4
7
10
64.5-86.6
74.0-82.0
67.1-84.4
73.1
77.7
76.5
Table 2.-Response
of Lymphocytes to
Stimulation with Native D N A
NO.
Studied
No&al Individuals
G-I Disease
Seizure Disorder
Cardiovascular Disease
Rheumatoid Arthritis
IIepatitis
Hepatitis
l’:orias s
SLE
% Increase
Large Cells
& Mitoses
Range
10
1
1
2
4
0-2
0
3
0
0-3
1
0
1
1
10
11
12
4-34
-
11 patients with other diseases also showed
no response to DNA. One of the two patients with hepatitis and one patient with
psoriasis and arthritis had 10 per cent and
11 per cent responses to DNA respectively.
Eleven of the 12 patients with SLE had a
positive response to DNA (Fig. 1). The
patient whose lymphocytes did not respond
RESULTS
significantly to DNA was being treated
Response in control tubes (tubes with with 80 mg. of prednisone daily. Other
no additive): The pattern of response-i.e.,
patients with SLE were taking 25 mg. or
numbers of large cells and cells in mito- less of prednisone. Lymphocytes were obsis-in tubes with no additive was similar tained from another patient with SLE who
in patients with SLE, patients with other was being treated with 60 mg. of preddiseases, and healthy normal individuals. nisone daily, but the lymphocytes failed
Response to PHA: The per cent of re- to grow in the control culture tubes so
sponse in tubes with PHA was similar that the response to DNA could not be
in patients with SLE, patients with other evaluated. Four patients had taken chlorodiseases, and healthy normal individuals quine for from three months to four years,
but this did not prevent the response of
(Table 1 ) .
Response to DNA: There was essentially their cells to DNA. The mean degree of
no response to DNA in the ten healthy response to DNA of lymphocytes from panormal individuals (Table 2). Nine of the tients with SLE was 12.6 per cent com-
34
PATRUCCO, ROTHFIELD AND HInSCHHOnN
30
X increase
Large cells
+
25
mitoses
20
15
10
5
...
..,
<
b
.........
......t
n
" -
I
2
3
4
5
6
7
8
9
10
II
12
L
mean
,,f21
controls
Fig. 1.-The
response of lymphocytes from patients with SLE to DNA. For comthe average of the response in the 21 other individuals tested is also shown.
The asterisk indicates the average of three experiments at monthly intervals using
lymphocytes from the same patient.
parison
DISCUSSION
pared to the mean of 1.6 per cent for the
21 others studied. The difference is statisIt is now well established that morphotically highly significant ( t test with Coch- logic changes and mitoses occur in cultured
rane's correction: p < .OOl).
peripheral blood lymphocytes as a response
Response to Denatured DNA: Cells of to specific antigens to which the donor is
control patients which did not respond to
native DNA did not respond to stimulaTable 3.-Response of Lymphocytes from
tion with denatured DNA (Table 3). The
Controls and Patients with SLE to
hepatitis patient who had a response to
Native and Denatured DNA
native DNA also responded to denatured
70Increase
Large Cells & Mitoses
DNA. Of the four patients with SLE tested,
N Tt 've
Denatured
the cells of three responded to both native
DNA
DNA
and denatured DNA, while the other reNormal
0
0
sponded only to native DNA.
Normal
0
0
Relation of response of lymphocytes to
Rheumatoid Arthritis
2
0
Rheumatoid Arthritis
1
2
DNA and titer of antinuclear antibody:
Rheumatoid Arthritis
0
0
There was no correlation between the titer
Hepatitis
10
10
of antinuclear antibody and the degree of
SLE
34
24
response to DNA in patients with SLE.
SLE
9
8
Antinuclear antibodies were not present
SLE"
F
11
SLE
13
3
in sera from the patients with other diseases or the healthy individuals (Fig. 2).
*Average result of one patient studied 3 times.
35
RESPONSE OF CULTURED LYMPHOCYTES
35
30
X increase
of large cells
+
25
mitoses
of tar
native DNA
15
0
IC
5
C i/
8
16
32
64
128
256
512
1024
I / A N F titer
Fig. 2.-The relation of titer of antinuclear antibody and degree of lymphocyte
response to stimulation with DNA.
sensitized.8J0 This response has been dem- immunologically competent cells are capaonstrated for such varied substances as ble of producing antibodies to more than
bacterial and viral extracts,1° antibiot- one nuclear antigen.17 The finding that
ics,11J2 and other allergens.1° Sensitivity lymphocytes from patients with SLE are
to allogeneic cells based on histocompati- sensitized to DNA is, therefore, not unexbility differences as well as to the patients’ pected, even when a low titer of antinucown skin cells in infantile excema have lear antibodies is present in the serum.
also been described.13J4 The latter find- The lymphocyte stimulation experiments
ing indicates that autoimmunity can be reflect the capacity of the cells to recogdemonstrated by this culture system, and nize the antigen, rather than the amount
has prompted us to study the response of of antibody that is currently being produced or circulating.
cells from patients with SLE to DNA.
The clear-cut response of the lymphoAntibodies to various nuclear antigens
have been reported by many investigators cytes from SLE patients to DNA and the
to occur in serum from patients with SLE. lack of the response in most other individThese nuclear antigens include the whole uals suggests a specific capacity of the
nucleus, nucleoprotein, DNA, histone, a cells to respond to DNA. Recent prelimiphosphate buffer nuclear extract, and a nary experiments using lymphocytes from
recently reported additional nuclear anti- other SLE patients have confirmed this
gen.3-6J5J6Antibodies to more than one finding and have, in addition, revealed that
of these nuclear antigens are commonly the lymphocytes from SLE patients negapresent in SLE sera, and it is likely that tive to PPD will not respond to another
36
PATRUCCO,
polyanion, purified lipopolysaccharide, or
to PPD, but do respond to DNA.
SLE lymphocytes responded not only to
native, but also to denatured DNA. Stollar
and Levine found that most SLE sera reacted more effectively with denatured DNA
than with native DNA.1s The positive reaction of lymphocytes from the patient with
psoriasis and arthritis remains to be explained. The patient did not have antinuclear antibodies nor did 16 of 17 other
patients with psoriasis and arthritis tested
in our l a b o r a t ~ r y .The
~ ~ positive test of
one of our two patients with actue hepatitis
may relate to the finding that LE cells
have been reported in this disorder.”J1
There was no spontaneous increase in
cellular enlargement or mitosis in cells from
L,E patients, beyond that found in normals,
at the time the cultures were harvested.
The findings by Cooper and Firkin that
there is an increased spontaneous rate of
mitosis and DNA incorporation during the
first hour of cluture reflect in vivo activity
of these cells.22 These active cells are no
longer found by the second day of culture
and are certainly no longer active by the
fifth day, when our assays were performed.
ROTHFIELD AND HIRSCHIIORN
The similarity of results in cultures without
stimulant in SLE patients and normals also
eliminates the possibility that the minute
amount of DNA in the culture medium.
released from dying cells, plays any role
in these results.
I n a recent study, it was shown that
there was perfect correlation between the
in vitro lymphocyte response and immediate wheal and flare reaction to penicillin
or penicilloyl antigens in patients with
clinical penicillin allergies.12 In that study,
there was no correlation between the in
vitro response and the titer of circulating
antipenicillin antibody of either IgG or
IgM type. There was no clear-cut correlation with delayed hypersensitivity. We
suggest that a positive response to DNA
by cells from patients with SLE may, as
with penicillin, be a sensitive indicator of
the presence of circulating cells previously
sensitized to this antigen.
ACKNOWLEDGhlENT
We wish to thank Dr. Beatriz Pogo of Rockefeller University for performing the DNA assays,
and Mrs. Maureen Toulon and h4iss llianca Pollo
for technical assistance.
SUMMARY
Lymphocytes from normal individuals and from most patients with other diseases
did not respond to stimulation with DNA in vitro by enlarging and dividing. Lymphocvtes from 11 of 12 patients with SLE showed a response to stimulation with DNA.
The response to heat-denatured DNA in controls and SLE patients was similar to the
response to native DNA. No relationship between degree of response of the SLE
lymphocytes to DNA and the titer of circulating antinuclear antibody could be
demonstrated.
IN INTERLINGUA
SUMMARIO
Lymphocytos ab subjectos normal e ab le majoritate de patieiites con morbos altere
clue disseminate lupus erythematose non respondeva per allargamento e division a1
stimulation in vitro con acido deoxyribonucleic. Lymphocytos ab 11 de 12 patientes con
disseminate lupus erythematose respondeva positivemente a stimulation con acido deoxyribonucleic. Le responsa a thermo-denaturate acido deoxyribonucleic in subjectos de
control0 e in patientes con disseminate lupus erythematose esseva simile a1 responsa a
native acido deoxyribonucleic. Nulle relation inter le grado del responsa del lymphocytos in disseminate lupus erythematose a acido deoxyribonucleic e le titro del circulante anticorpore antinucleari poteva esser demonstrate.
37
RESPONSE OF CULTURED LYMPHOCYTES
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