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Deficient ╨Ю╤Ц-interferon production in patients with systemic lupus erythematosus.

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1210
DEFICIENT 7-INTERFERON PRODUCTION
IN PATIENTS WITH
SYSTEMIC LUPUS ERYTHEMATOSUS
GEORGE C. TSOKOS, DIMITRI T. BOUMPAS, PAULA L. SMITH, JULIE Y. DJEU,
JAMES E. BALOW, and ALAIN H. ROOK
We examined the ability of peripheral mononuulear cells (MNC) from patients with systemic lupus
erflhematosus (SLE) to produce y-interferon (y-IFN) in
vitro. MNC from patients with SLE produced varying
amounts of y-IFN upon mitogenic stimulation. However, they produced distinctly decreased amounts of
y-WN upon in vitro stimulation with interleukin-2
(IL-2). Deficient production seemed to be primary,
rather than secondary to either excessive monocytic
suppresion or failure of IL-2 to bind to the MNC surface
membranes. y-IFN-specific RNA transcription, as determined by slot-blot analysis, was severely decreased in
MNC that had been stimulated with phytohemagglutinin or IL-2. These findings suggest that MNC of
patients with SLE have defects in the IL-2 signal
transduction which is required for production of y-IFN.
Systemic lupus erythematosus (SLE) is the
prototype human autoimmune disease and is characterized by multiple abnormalities of both humoral and
cellular immune responses (1). Cellular cytotoxic re--From the National Institutes of Health, Bethesda, Maryland.
George C. Tsokos, MD: Kidney Disease Section, National
Institute of Diabetes, Digestive and Kidney Diseases, NIH; Dimitri
T. Boumpas, MD: Kidney Disease Section, National Institute of
Diabetes, Digestive and Kidney Diseases, NIH; Paula L. Smith, BS:
Kidney Disease Section, National Institute of Diabetes, Digestive
and Kidney Diseases, NIH; Julie Y. Djeu, MD: Department of
Microbiology, University of South Florida, Tampa; James E.
Balow, MD: Kidney Disease Section, National Institute of Diabetes, Digestive and Kidney Diseases, NIH; Alain H. Rook, MD:
Laboratory of Immunoregulation, National Institute of Allergy and
Infectious Diseases, NIH.
Address reprint requests to George C. Tsokos, MD, NIH,
Building 10, Room 3N-114, Bethesda, MD 20892.
Submitted for publication October 7, 1985; accepted in
revised form May 13, 1986.
Arthritis and Rheumatism, Vol. 29, No. 10 (October 1986)
sponses consistently have been reported to be abnormally low, with defects reported in antibody-dependent cellular cytotoxicity (2), cytotoxic responses
against xenoantigens (3) and alloantigens (4), and
natural cytotoxicity (5-7). Interleukin-2 (IL-2) and
yinterferon (yIFN) are known to participate in the
expression of cytotoxic activity by mononuclear cells
(MNC). yIFN, as well as several of its known inducers, augment natural killer (NK) cell activity (8,9);
IL-2 sustains cytotoxic T cell lines (lo), enhances NK
cell activity (1l), supports proliferative responses (12),
modulates the function of the immunoregulatory cells
(13), and enhances allogeneic cytotoxic responses
(14). Both IL-2 (15,16) and y I F N (6,17) production in
vitro have been noted to be deficient in certain patients
with SLE.
IL-2 has been recognized to affect several cellmediated immunologic reactions (18-21), some of
which may involve interaction with yIFN. Since IL-2
can induce the production of y I F N by normal peripheral MNC in vitro (22), we investigated the ability of
IL-2 to induce y I F N production by peripheral MNC
from SLE patients. Neither IL-2 nor phytohemagglutinin (PHA) was able to induce y I F N production at
levels comparable with those produced by normal
MNC. Levels of yIFN-specific RNA were markedly
decreased in parallel, in MNC from patients with SLE.
PATIENTS AND METHODS
Patient population. Twenty-nine patients who fulfilled the American Rheumatism Association criteria for the
classification of SLE (23) and were observed at the Arthritis
and Rheumatism Branch of the National Institute of Arthritis
and Musculoskeletal Diseases were included in this study.
Disease activity was defined by the presence of clinical cri-
y-IFN IN SLE
teria (i.e., arthritis, serositis, glomerulonephritis, central
nervous system involvement, cytopenia) and serologic criteria (i.e., low serum C3 levels and elevated serum DNA
binding). A patient was considered to have active disease if
1 of the clinical and 1 of the serologic criteria were present.
Four of the patients with active disease were not
receiving treatment. Seventeen were taking prednisone (a
maximum of 40 mg daily), which was withheld 24 hours prior
to blood sampling, and 4 patients had received cyclophosphamide 1 or more months prior to blood sampling. Four
more patients had inactive disease; they were treated with
low doses of prednisone. Healthy age- and sex-matched
volunteers served as controls.
Mononuclear cell preparation. MNC were obtained
by standard Ficoll-Hypaque gradient centrifugation. Patient
and control MNC were thawed after they had been stored in
liquid nitrogen. Cells were frozen in RPMI 1640 (Gibco,
Grand Island, NY) which contained 10% DMSO (Sigma, St.
Louis, MO) and 20% fetal calf serum (FCS; Gibco). (Responses of MNC from normal individuals and from patients
with SLE are not affected by this freezing and thawing
procedure; data not shown.) Lymphocyte subsets defined by
reaction with commercial monoclonal antibodies against T
helper and suppressor cells were comparable in MNC from
patients and from controls (unpublished data).
y-IFN production. In order to study the capacity of
peripheral MNC to produce y-IFN, the MNC of the study
subjects were suspended at a concentration of 2.5 x lo6
cells/ml in RPMI 1640 medium supplemented with 10% FCS
and then were incubated with medium, phytohemagglutinin
(PHA, 1 kg/ml; Burroughs-Wellcome, Beckenham, UK), or
recombinant IL-2 (Cetus, Emoryville, CA). In preliminary
studies, maximal production of yIFN in response to the
above stimuli occurred between 48 and 72 hours. Therefore,
after a 60-hour incubation at 37°C in 5% C02 in air, the cell
suspensions were centrifuged, and the supernatant fluids
were maintained at -70°C until assayed.
y-IFN assay. Antiviral activity was determined by the
reduction of vesicular stomatitis virus plaque formation on
human amnion (WISH) cells (ATCC CCL25) grown in
microtiter plates (22). The antiviral activity, expressed in
IFN units, was calculated as the reciprocal of the highest
dilution of the sample that reduced the number of viral
plaques by 50%.
RNA extraction and hybridization. RNA was isolated
from cells by guanidine thiocyanate extraction and pelleting
through cesium chloride by centrifugation (24). Slot-blot
analysis of y-IFN-specific RNA was performed using a
previously described minifold device (25). Serial threefold
dilutions of the RNA samples (3 pg, 1 pg, and 0.33 wg) were
denatured in 2.2M formaldehyde/l5~SSC (1 xSSC =
0.015M sodium citrate plus 0.15M sodium chloride, pH 7) for
15 minutes at 68"C, transferred onto nitrocellulose filters
using the slot-blot apparatus, and hybridized to the 32P-nicktranslated y I F N complementary DNA (cDNA) probe
(kindly provided by Dr. S. Arya, National Cancer Institute).
All RNAs were examined following denaturation for degradation by agarose gel electrophoresis. None showed signs of
degradation. Probe DNA was labeled with a 32P-dCTPto
approximately 1 x lo8 counts per minute/pg. RNA extracted
from normal human T cells stimulated with PHA and normal
121 1
human bronchial epithelial cells were used as positive and
negative controls. Filters were exposed to Kodak (Rochester, NY) XAR-5 films for 36 hours. Films were subjected to
densitometric measurement using the Beckman (Palo Alto,
CA) DU-8 spectrophotometer.
RESULTS
y-IFN production in response to PHA. In order
to investigate the ability of MNC from SLE patients to
produce y I F N , we used PHA to stimulate MNC from
9 patients with active disease and 4 patients with
inactive disease. MNC from normal individuals (n = 7)
produced 352 k 52 units/ml (mean & SEM) of y I F N .
MNC from patients with active disease produced 97 ?
30 unitdm1 (P < 0.02), while those from patients with
inactive disease produced 151 2 35 units/ml (P <
0.02). Although MNC from SLE patients as a group
produced less y-IFN than did MNC from normal
individuals upon mitogenic stimulation, a subgroup of
patients, some with active disease and some with
inactive disease, exhibited responses comparable with
those of normal individuals (Figure 1).
y-IFN production in response to IL-2. We next
investigated the ability of IL-2 to induce y I F N production by peripheral MNC from patients with SLE.
Figure 2 shows the levels of y I F N production, upon
stimulation with 25 units/ml or 100 units/ml of 1L-2, by
MNC from 25 patients with active disease (4 receiving
no treatment and 21 receiving various doses of
prednisone), 4 patients with inactive disease, and 8
normal individuals. A concentration of 100 unitdm1 of
IL-2 induced the maximum release of y I F N from
normal MNC (22). MNC from patients with active
disease produced 26 ? 8 units/ml (mean & SEM),
those from patients with inactive disease produced 600
t 336 units/ml, and those from normal individuals
released 693 t 232 unitdml. These data show clearly
that y I F N production is severely depressed in patients with SLE, upon in vitro stimulation of their
MNC with IL-2.
Higher (up to 500 units/ml) and lower (down to
0.1 unitdml) concentrations of IL-2 were used in
certain experiments, but this did not result in correction of the deficient y I F N release (Table 1). When
double stimulation, with 1 pg/ml of PHA and 100
unitdm1 of IL-2, was employed, the response was
virtually similar to that obtained with PHA alone.
MNC from patients with SLE and from normal
individuals were depleted of monocytes by passage
through Sephadex G-10 columns (4), in order to investigate their possible suppressive role in the production
TSOKOS ET AL
1212
0
0
0
0
0
0
-6
0
0
8
0
-8-
0
0
0
- e - m - e Y o
0
0
0
C
D
O
0
0
0
0
I
0
0
0
CD
0
CD
+%3
0
0
0
0
1
IL2 (Unitdml) 25
100
Active
PHA
Figure 1. Phytohemagglutinin (PHA) stimulation of y-interferon
production by mononuclear cells from patients with active and
inaative systemic lupus erythematosus and from normal individuals.
of yIFN. This procedure reduced the percentage of
M3+ (monocytic marker) cells by more than 95%.
y I F N production remained unchanged in MNC from 3
normal individuals and from 12 patients with SLE
(Table 2). Similarly, the presence of 2 pg/ml of
indomethacin (Sigma) did not affect y I F N production
(dcdta not shown).
yIFN RNA expression in response to IL-2 and
PEA. To determine whether the decreased production
of y I F N by MNC from SLE patients in response to
IL-2 and PHA was due to decreased transcription of
y IFN-specific DNA, we performed the following
experiment: MNC from 3 normal individuals and 3
patients with SLE (found to have decreased production of yIFN) were incubated for 24 hours in the
prasence of 100 units/ml of IL-2 or 1 pg/ml of PHA.
RNA was extracted from these cells and hybridized to
32P-nick-translated y I F N cDNA probe. Patients with
SLE showed a major impairment in the appearance
of yIFN-specific RNA levels in response to IL-2
or PHA; the response in normal individuals was at
I
25
100
Inactive
25
100
Normal
Figure 2. Interleukin-2 (IL2) stimulation of ?interferon production
by mononuclear cells from patients with active and inactive systemic lupus erythematosus and from normal individuals.
least ninefold higher, by densitometer measurement
(Figure 3). Moreover, in contrast to normal MNC,
MNC from SLE patients showed a complete absence
of yIFN-specific messenger RNA after stimulation
with IL-2.
Table 1. y-interferon production (mean geometric titer), in the
presence of interleukin-2 (IL-2) andor phytohemagglutinin (PHA),
by mononuclear cells from patients with active systemic lupus
erythematosus and from normal controls
Stimulus
Patients
(n = 3)
Normal controls
(n = 3)
IL-2 (unitshl)
0
0.1
0.5
1.o
5.0
10.0
100.0
500.0
1 p g h l PHA
100.0
0.0 + 1 p g h l PHA
2
5
12
30
25
25
30
60
250
250
19
25
30
75
126
250
625
625
925
925
+
y-IFN IN SLE
1213
Table 2. y-interferon production (geometric titer, mean 2 SEM),
after monocyte depletion, by mononuclear cells (MNC) from
patients with active systemic lupus erythematosus and from
normal controls
Patients
(n = 12)
Normal controls
(n = 3)
Stimulus*
MNC
G-10 cellst
MNC
G-10 cellst
0
<5
<5
<5
<5
17 5
82 18
<5
110 35
530 f 180
620 f 230
<5
115 k 43
525 170
680 k 190
25 units IL-2
100 units IL-2
1 pg PHA
15 2 4
75 f 21
*
*
*
* Final concentration per mlof culture medium. IL-2 = interleukin-2;
PHA = phytohemagglutinin.
t MNC depleted of monocytes by passage through G-10 column.
DISCUSSION
Our results confirm that the production of yIFN by peripheral MNC from patients with SLE, upon
stimulation by mitogens in vitro, is deficient. Mitogens
appear to b e potent stimuli, and the amounts of y-IFN
produced by MNC from patients with SLE span a
broad range. This study, as well as previous ones
(6,17), demonstrate that only a portion of patients with
SLE exhibit a deficient response following mitogenic
triggering of MNC. Similarly, the production of IL-2
by MNC in response to PHA is diminished in only a
small subgroup of patients with clinically active SLE
(ref. 17 and unpublished data).
In contrast with the results obtained with PHA,
the use of more subtle stimuli (such as IL-2 in the
present experiments) or the use of viral inducers (26)
results in distinctly depressed y I F N release by MNC
from SLE patients, in comparison with normal subjects. Thus, this study demonstrates that IL-2 fails to
induce production or release of y I F N by MNC from
patients with active SLE. Moreover, our results indicate that this deficiency seems to be an intrinsic
cellular defect, rather than secondary to excessive
suppressor cell activity. Monocytes have been found
to be responsible, at least in part, for several deficient
in vitro T and B lymphocyte responses of MNC from
patients with SLE (1); however, they were excluded as
a cause of suppression of y I F N production. Other
cellular sources of suppression were not investigated
in this study.
Figure 3. Hybridization of the y-interferon complementary DNA clone probe to the total
cellular RNA extracted from mononuclear cells (MNC) from a normal individual and from a
patient with systemic lupus erythematosus (SLE). MNC were cultured in the presence of either
medium, interleukin-2 (IL-2), or phytohemagglutinin (PHA). Controls included cellular RNA
extracted from normal human bronchial epithelial cells (NHBE) and T lymphocytes stimulated
with PHA. Filters were exposed for 36 hours.
1214
TSOKOS ET AL
We have previously shown that IL-2 can consistently augment the diminished allogeneic cytotoxic
and NK cell responses of MNC of SLE patients in
vitfo (21). Previously, our explanation for this enhancement would have included the induction of y
IFW, since it can enhance cytotoxic response (27,28)
and IL-2 induces the release of y I F N by normal MNC
(22). However, findings in the present study suggest
that the IL-2-induced enhancement of cytotoxic responses is not the result of augmented y I F N release.
The possibility that deficient y I F N production by
MNC from patients with SLE is secondary to the
faihre of IL-2 to bind to its surface receptor can be
excluded by the fact that IL-2 can enhance the cytotoxic responses of these cells (21). Furthermore, MNC
from patients with SLE express normal levels of IL-2
receptors after stimulation with PHA (unpublished
observations).
The failure of IL-2 and PHA to induce the
expression of yIFN-specific RNA in MNC from SLE
patients indicates that these cells do not transcribe the
DNA message to RNA. Alternatively, y I F N RNA
degradation might be responsible for the observed
decreased message. This could be secondary to either
faihre of transmission of the signal to the nucleus or to
primary transcriptional abnormality. Further experiments are needed to resolve this question.
In conclusion, y I F N production and release by
MNC from patients with SLE, and particularly from
thase with active disease, are severely compromised.
Since y I F N participates in the expression of various
lymphocyte functions, detailed investigation of the
role of deficient y I F N production in the expression of
multiple cellular abnormalities in SLE patients is important.
ACKNOWLEDGMENTS
We wish to thank Dr. S. Arya of the National Cancer
Institute for providing the y I F N probe, and Linda Adams
for her help in the preparation of this manuscript.
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