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In vivo production of interleukin-10 by nont cells in rheumatoid arthritis sjugren's syndrome and systemic lupus erythematosus.

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ARTHRITIS & RHEUMATISM Volume 37
Number 11, November 1994, pp 1647-1655
0 1994, American College of Rheumatology
1647
IN VIVO PRODUCTION OF INTERLEUKIN-10 BY NON-T CELLS IN
RHEUMATOID ARTHRITIS, SJOGREN’S SYNDROME, AND
SYSTEMIC LUPUS ERYTHEMATOSUS
A Potential Mechanism of B Lymphocyte Hyperactivity and Autoimmunity
LUIS LLORENTE, YVONNE RICHAUD-PATIN, RENATO FIOR, JORGE ALCOCER-VARELA,
JOHN WIJDENES, BRIGITTE MOREL FOURRIER, PIERRE GALANAUD, and DOMINIQUE EMILIE
Objective. Interleukin-10 (IL-10) is a potent stimulator of B lymphocytes in vitro. In vivo dysregulation
of IL-10 gene expression was therefore analyzed in
patients with rheumatoid arthritis (RA), primary Sjogren’s syndrome (SS), and systemic lupus erythematosus (SLE).
Methods. Spontaneous production of IL-10 by
peripheral blood mononuclear cells was measured using
reverse transcription polymerase chain reaction and
enzyme-linked immunosorbent assay in untreated patients with either RA (n = lo), SS (n = lo), or SLE
(n = lo), and in 15 normal control subjects.
Results. IL-10 production was dramatically
higher in RA, SS, and SLE patients than in controls. In
each group, both B lymphocytes and monocytes, but not
T lymphocytes, produced IL-10.
Conclusion. IL-10 production is increased in RA,
SS, and SLE. It may play a role in B lymphocyte
hyperactivity and in the development of autoimmunity.
Supported in part by grants from the Commission of the
European Communities (CI1*-CT92-W5), the Consejo Nacional de
Ciencia y Tecnologfa (CONACYT) (21226-5-1626M), Mexico, and
the Association pour la Recherche sur la Polyarthrite, France.
Luis Llorente, MD: Instituto Nacional de la Nutrici6n
Salvador Zubirfin, Mexico City, Mexico; Yvonne Richaud-Patin,
BS: Instituto Nacional de la Nutrici6n Salvador Zubirfin; Renato
Fior, MD: U.131 de 1’Institut National de la SantB et de la Recherche MBdicale (INSERM), Clamart, France; Jorge AlcocerVarela, MD: Instituto Nacional de la Nutrici6n Salvador Zubildn;
John Wijdenes, PhD: Innothkrapie, Besancon, France; Brigitte
Morel Fourrier, PhD: InnothBrapie; Pierre Galanaud, MD:
INSERM U.131; Dominique Emilie, MD, PhD: INSERM U.131.
Address reprint requests to Luis Llorente, MD, Department of Immunology and Rheumatology, Instituto Nacional de la
Nutrici6n Salvador Zubirhn, Vasco de Quiroga 15, 14000 Mbxico,
D.F., MBxico.
Submitted for publication January 25, 1994; accepted in
revised form June 14, 1994.
Several human rheumatic autoimmune diseases, such as rheumatoid arthritis (RA), Sjogren’s
syndrome (SS), and systemic lupus erythematosus
(SLE), are characterized by a prominent B lymphocyte hyperactivity that results in both an increased
production of immunoglobulins and the emergence of
autoantibodies. This hyperactivity appears to play a
role in the development of clinical manifestations,
either by deposition of immune complexes or by
recognition of self antigens.
The mechanism by which B lymphocytes remain inappropriately activated for years in patients
with rheumatic autoimmune diseases is still poorly
understood, and may involve several agents acting in
concert: genetic background (l), environmental factors such as drugs (2), hormonal influences (3), viruses
(4-7), and abnormal production of cytokines. With
respect to the latter, most studies have been performed in RA patients, and have shown that several
cytokines with the potential to activate B lymphocytes, such as interleukin-1 (IL-1), tumor necrosis
factor a (TNFa), and IL-6, are produced within inflamed joints and are present in abnormally increased
amounts in the serum (8-1 1). Some of these cytokines
have been shown to augment destructive cellular activities in RA (12-16). It has recently been shown that
treatment of RA patients with an anti-TNFa antibody
improves the clinical course of the disease (17).
To our knowledge, no study thus far has demonstrated a dysregulation of cytokine production in SS
patients. An increased level of IL-6 has been detected
in the serum of patients with active SLE. Although
peripheral blood mononuclear cells (PBMC) from SLE
patients produce decreased amounts of this cytokine
LLORENTE ET AL
1648
upon stimulation, B lymphocytes from SLE patients
spontaneously produce IL-6 and constitutively express IL-6 receptors (18-21). In vitro inhibition of this
autocrine loop by anti-IL-6 receptor antibodies decreases the spontaneous production of autoantibodies
(2 1). Anti-TNFa antibodies also decrease the production of immunoglobulins by cultured PBMC obtained
from SLE patients (22), but circulating TNFa is not
decreased in such patients (23). Since no unifying
abnormality of cytokine regulation has been evidenced
in rheumatic autoimmune diseases, it is possible that
another yet-uncharacterized cytokine contributes to B
lymphocyte hyperactivity in these conditions.
It has recently been demonstrated that IL-10 is
one of the most potent activators of B lymphocytes,
inducing in vitro both their proliferation and a strong
production of immunoglobulins (24). Moreover, IL-10
may prolong B lymphocyte survival by inducing bcl-2
production by these cells, thus protecting them from
programmed cell death (25). IL-10 may also play a role
in the emergence of B lymphomas, particularly in the
context of abnormal Epstein-Ban virus (EBV) replication (26-28).
With these properties in mind, we sought to
determine whether a dysregulation of IL-10 production in vivo may provide a common mechanism leading to sustained B lymphocyte hyperactivity in RA,
SS, and SLE. We studied the spontaneous production
of this cytokine by freshly isolated PBMC and observed a profound abnormality of IL-10 gene regulation in each of these diseases.
PATIENTS AND METHODS
Patient selection. We studied 30 patients with rheumatic autoimmune diseases who were receiving no medication and had not received corticosteroids for at least l
month, or immunosuppressors, gold salts, D-penicillamine,
or antimalarials for at least 3 months, or nonsteroidal antiinflammatory agents for at least 5 days. Ten patients had
SLE, fulfilling at least 4 of the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) 1982 revised criteria for SLE (29), 10 patients had
RA, fulfilling the ACR 1987 criteria for RA (30), and 10
patients had satisfied the diagnostic criteria for primary SS (31).
SLE disease activity was graded according to a
previously published index (32). This index was used to
designate the patients as having inactive disease, mildly
active disease, moderately severe disease, and severe multisystem disease. Patients with RA were considered to have
active disease if they presented with 6 or more swollen joints
and at least 2 of the following: 29 joints tender to pressure,
2 1 hour of morning stiffness, and a Westergren erythrocyte
sedimentation rate 228 mrdhour. Disease activity or inac-
tivity is usually less clearly apparent in patients with primary
SS; thus, patients were not subcategorized.
Fifteen healthy volunteers were studied as controls.
Another 17 patients who were seropositive for human immunodeficiency virus type 1 (HIV-1) with a peripheral
CD4+ T cell cell count >200/pl and without opportunistic
infection or malignancy were also studied as controls. All
study subjects were informed about the objectives and
methods of the study and gave their consent.
Purification and culture of PBMC. PBMC were isolated on Histopaque (Sigma, St. Louis, MO) by gradient
centrifugation. After 2 washings, cells were enumerated, and
pellets of 10 million cells were immediately frozen at -70°C.
Cell subpopulations were purified as follows: PBMC were
cultured for 2 hours on plastic petri dishes. Adherent cells
were recovered using a rubber policeman after 1 hour at 4°C
in phosphate buffered saline (PBS) containing EDTA. This
population contained >85% CD14+ cells as determined by
flow cytometry analysis.
T cells and B cells were isolated from the nonadherent cell population by rosetting twice with AET (Sigma)treated sheep red blood cells (SRBC). AET-treated SRBC
rosetting cells (T cells) were >97% CD3+ cells. B cell
preparations contained >92% CD20+ cells. One million
unfractionated PBMC or cell subpopulations were cultured
for 24 hours in 1 ml of 10% gamma globulin-free fetal calf
serum (FCS) (Gibco-BRL, Gaithersburg, MD) containing
RPMI 1640 medium. The concentration of IL-10 was then
measured in the supernatant by enzyme-linked immunosorbent assay (ELISA). The same lot of FCS was used throughout all experiments.
Detection of IL-10 messenger RNA (mRNA) by semiquantitative reverse transcription polymerase chain reaction
(RT-PCR).Total cellular RNA was extracted from 10 million
frozen cells using the RNAzol technique according to the
manufacturer’s recommendation (Biotecx, Houston, TX).
The RNA was treated at 37°C for 30 minutes with 10 units of
RNase-free DNase (Gibco-BRL), then extracted with phenol
and chlorofordisoamylic alcohol (24/1 volume/volume), and
precipitated with ethanol. RNA was then quantified by
measuring the optical density at 260 nm.
RT-PCR was performed using 2 pg of RNA that had
been treated for 1 hour at 42°C with 50 units of Moloney
murine leukemia virus reverse transcriptase (Gibco-BRL) in
the presence of 10 pmoles oligo(d[T],,-,,) (Gibco-BRL) and
0.5 mM of each of the 4 dNTP. For each sample, half of the
RT product was then processed for IL-10 PCR.
To the reaction mixture was added 30 pmoles antisense primer (5‘-AGAGCGCCAGATCCGATTTT-3’), 30
pmoles sense primer (5’-ATCAAGGCGCATGTGAACTC37, and 2 units of Taq polymerase (Gibco-BRL). The oligonucleotides we used are specific for the human IL-10 gene
and do not co-amplify the BCRFl gene of EBV. The expected
size of the amplified product is 295 basepairs. Each cycle of
amplification was at 94°C for 1 minute, 55°C for 1 minute, and
72°C for 1 minute. The products of 28 cycles were subjected to
electrophoresis on agarose gels and stained with ethidium
bromide. In 8 samples, Southern blot analysis verified that
the amplified product was recognized by an internal IL-10
32P-labeled oligonucleotide (5’-CATCGATTTCTTCCCTGTGA-3’).
IL-10 IN RHEUMATIC AUTOIMMUNE DISEASES
1649
Figure 1. Interleukin-10 (IL-10) and pactin messenger RNA (mRNA) amplification in autoimmune rheumatic diseases. Reverse transcription polymerase chain reaction was used to
amplify mRNA coding for IL-10 or for pactin from peripheral blood mononuclear cells
obtained from 15 healthy individuals, 10 patients with rheumatoid arthritis (RA), 10 with
Sjogren’s syndrome (SS), and 10 with systemic lupus erythematosus (SLE). Shown are
representative results of ethidium bromide staining from 1 experiment. Lanes a-1, Amplified
pactin and IL-10 from 2 patients with SS, 2 with SLE, and 2 with RA, respectively; lanes m-p,
amplified pactin and IL-10 from 2 healthy individuals;far left lane, phage X-174 DNA digested
with HaelII.
The other half of the RT product was screened for
pactin mRNA using the same procedure with the following
oligonucleotides: 5’-GGTCTCAAACATGATCTGGG-3’
and 5’-GGGTCAGAAGGATTCCTATG-3’as antisense and
sense oligonucleotides, respectively. The size of the amplified &actin product was 268 bp. For both IL-10 and p-actin
RT-PCR, samples from patients and controls were processed
in parallel. Negative controls included samples in which the
addition of RT was omitted during the processing, and
samples in which no complementary DNA was added during
the PCR reaction.
The intensity of the band for IL-10 and pactin
RT-PCR products was determined by densitometry (Hoefer
Scientific Instruments, San Francisco, CA). We verified that
the number of cycles used for IL-10 and p-actin PCR (28
cycles) is suboptimal for PCR amplification, i.e., that the
plateau level for PCR product is not reached at the end of the
reaction. Results are expressed for each sample as the ratio
between IL-10 and p-actin band intensity.
ELISA for 1L-10. IL-10 levels were determined by
ELISA. Anti-IL-10 monoclonal antibody (MAb) B-NlO was
used as the coating antibody, and anti-IL-10 MAb B-TI0 as
the tracer antibody. The sensitivity of this ELISA is 3 pg/ml,
and it does not recognize the BCRFl gene product.
Each well was coated overnight with 100 pl of MAb
B-N10 (2.5 p g h l in 0.05M bicarbonate buffer) at 4°C. The
plate was washed once with washing buffer (water containing 0.05% Tween 20), incubated with 250 pl/well of blocking
buffer (20% sucrose in 0.1MTris, pH 7.7) for 2 hours at 20”C,
and then the buffer was aspirated and discarded. To each
well was added 100 pl of standards and samples diluted in
sample buffer (PBS, 1% bovine serum albumin [BSA], 0.1%
thimerosal) and 50 pywell of biotinylated B-T10 (500 ng/ml in
PBS, 1% BSA, 3% normal mouse serum, 0.1% thimerosal).
The plates were incubated for 2 hours at 37°C. The wells
were washed 4 times between each subsequent step. One
hundred microliters of streptavidin peroxidase (1 pg/ml in
sample buffer; Sigma) was added to each well and incubated
for 45 minutes at 20°C. After the last wash, 100 pl of 0.4
mg/ml o-phenylenediamine dihydroxychloride (Sigma) was
added to each well, and the plates were incubated for 30
minutes at 20°C. The enzyme reaction was stopped by
adding 100 pywell of 1N H2S04. Optical densities were
measured at 492 nm.
RESULTS
In vivo expression of the IL-10 gene by PBMC
from RA, SS, and SLE patients. Spontaneous expression of the IL-10 gene by circulating cells was evaluated by RT-PCR studies of freshly isolated PBMC.
This analysis was performed in parallel on cells from
RA, SS, and SLE patients and from normal control
subjects. Following amplification, a faint band was
detected in samples from 9 normal controls; no signal
was detected in samples from the other 6 subjects. In
contrast, positive results, in most cases of strong
intensity, were obtained in all samples from RA, SS,
and SLE patients. A representative experiment is
shown in Figure 1. The size of the amplified product
was as expected for IL-10 mRNA (295 bp). We verified
that the amplified product was recognized by an IL10-specific oligonucleotide (data not shown). p-actin
mRNA was detected in all patient and control samples
(data not shown).
To more accurately quantify the increased
IL-10 gene expression in RA, SS, and SLE patients as
compared with the controls, we determined for each
sample the ratio of the band intensity between IL-10
LLORENTE ET AL
1650
and P-actin RT-PCR products. We first determined the
relationship between tbis ratio and the amount of
IL-10 mRNA present. The band intensities for the
pactin mRNA amplified product from the undiluted
RNA sample and from graded dilutions of the same
sample were measured in 3 patients with high levels of
IL-10 rnRNA. Results from this experiment (Figure
2A) showed that a 90% decrease in IL-10 mRNA
concentration led to a 28% decrease in the IL-1O:p
actin ratio. This experiment also shows that the IL-10:
pactin ratio is directly related to IL-10 mRNA concentrations for ratios below 1.5.
We then determined the IL-1O:P-actin ratio for
all samples (Figure 2B). The mean ? SEM ratio was
0.22 & 0.06 in controls, and 0.98 -+ 0.13, 1.30 -+ 0.21,
and 1.16 5 0.13 in RA, SS, and SLE patients, respectively (P < 0.005 for each value versus controls, by
Mann-Whitney U test). From the relationship established in Figure 2A, this means that the average
amount of IL-10 mRNA in PBMC from RA, SS, and
SLE patients is -6.75-fold, S f o l d , and 10.3-fold
higher than in controls, respectively.
In vitro production of IL-10. To demonstrate
that the spontaneous expression of the IL-10 gene was
associated with a parallel increase in IL-10 production,
we measured IL-10 concentrations in the supernatants
of PBMC cultured for 24 hours without deliberate
stimulation. Very high amounts of IL-10 were detected
in samples from RA, SS, and SLE patients; only low
levels of IL-10 were spontaneously produced by
PBMC from normal subjects (P < 0.001 versus each
patient group, by Mann-Whitney U test) (Figure 3).
We next sought to determine whether increased
production of IL-10 is a general feature of diseases
characterized by B cell hyperactivity. To this end, we
measured spontaneous IL-10 production by PBMC
from HIV-infected patients who did not have profound
immune deficiency. Although production of IL-10 by
these patients was higher than that of the normal
controls, this increase was moderate (Figure 3) and
was significantly lower than that of the RA (P <
O.OOS), SS (P < 0.004), and SLE (P < 0.003) patients.
Correlation between in vivo IL-10 mRNA levels
and spontaneous in vitro production. Next, we determined whether the spontaneous IL-10 production
measured in short-term cultures truly reflected in vivo
IL-10 gene expression. To this end, we compared in
each sample the amount of IL-10 produced in vitro and
the IL-10 mRNA:pactin mRNA ratio determined in
freshly isolated cells (see Figure 1). Both parameters
A
s
c
20
-
a
z
E
*I
15-
2
1 0 -
0
.5
c
I
-
A
05
001
1
2
8
I
16
32
64
REClPROCAL OF RNA DILUTION
4
128
B
1
3.0
-
2.0
-
1.0
--
0
G
a
i
Y
-
0
t
7
*me
0.
Figure 2. Quantification of IL-10 mRNA in RA, SS, and SLE
patients. A, The relationship between the amount of IL-10 mRNA
and the 1L-lO:pactin mRNA ratio was determined in 2 SS patients
and 1 SLE patient who had high IL-10 mRNA levels in peripheral
blood mononuclear cells. The band intensity for the pactin mRNA
product of reverse transcription polymerase chain reaction was
determined from the undiluted RNA sample; that for the IL-10
mRNA product was determined from undiluted and serial dilutions
(from 1:2 to 1:128) of RNA samples. The ratio between the intensity
of the IL-10 band for a dilution and the intensity of the pactin band
for the undiluted sample was determined. B,The IL-1O:pactin ratio
was determined from undiluted complementary DNA samples from
healthy control subjects and from patients with RA, SS, or SLE. See
Figure 1 for other definitions.
165 1
IL- 10 IN RHEUMATIC AUTOIMMUNE DISEASES
were highly correlated (P< 0.001, by Spearman’s test)
(Figure 4). Taken together, these results indicate that
at both the mRNA and the protein level, there is
increased IL-10 gene expression by circulating cells in
RA, SS, and SLE patients compared with normal
subjects.
Absence of correlation between IL-10 production
and clinical or biologic markers of disease activity. The
level of spontaneous in vitro IL-10 production was first
compared with biologic markers of disease activity
such as autoantibody titers and prevalence of hypergammaglobulinemia. There was no correlation between IL- 10 production and titers of anti-doublestranded DNA (for SLE samples), anti-Ro and anti-La
(for SS samples), or rheumatoid factor and hypergammaglobulinemia (for SS and RA samples) (data not
shown). There was also no correlation in RA and SLE
patients between IL-10 production and clinical indices
of disease activity (data not shown). Although study of
larger series of patients is required to confirm these
findings, it appears that increased production of IL-10
by PBMC is a characteristic of rheumatic autoimmune
diseases regardless of the clinical and biologic activity
of the disease.
I
7000
6000 -
T
5000 -
1
2
a
4000
-
3000
-
2000
-
1000 -
0-
CONT. SLE
SS
RA
HIV+
Figure 3. Production of IL-10 by peripheral blood mononuclear
cells from RA (n = lo), S S (n = lo), and SLE (n = 10) patients. Cells
were cultured without stimulation for 24 hours, and the IL-I0
concentration in the supernatant was determined. Results were
compared with IL-10 production from either normal subjects (cont.)
(n = 15) or patients infected with the human immunodeficiency virus
(HIV+) (n = 17). See Figure 1 for other definitions.
3’0
2.5
z
-c
i-
0
0
c
c
I
=!
R=
-0.5
o
2000
4000
6000
0.86
8000 IOOOO 12000 14000
IL- t 0 pg/ml
Figure 4. Correlation between in vivo IL-I0 mRNA levels and in
vitro production. The relationship between in vivo IL-10 mRNA
levels in peripheral blood mononuclear cells (expressed as the
IL-1O:pactin ratio) and the spontaneous production of IL-10 in vitro
was determined for 15 healthy subjects and 10 patients each with
RA, SS,or SLE. See Figure 1 for definitions.
IL-10 arises from B lymphocytes and monocytes
in RA, SS, and SLE. To determine which cell population contributes to the increased IL-10 production in
RA, SS, and SLE, PBMC were fractionated, and
spontaneous in vitro production of IL-10 by purified T
lymphocytes, B lymphocytes, and macrophages was
assessed. The results are shown in Table 1.
The first point to emerge was that in most cases,
T lymphocytes did not contribute significantly to the
increased production of IL-10. Indeed, in almost every
patient, the IL-10 concentration in supernatants of
cultured T lymphocytes was dramatically lower than
that in supernatants of B lymphocytes or macrophages. The respective involvement of these 2 latter
cell subpopulations in IL-10 production varied from
patient to patient. Monocytes produced higher mean
levels of IL-10 than did B lymphocytes. However, the
opposite pattern was occasionally seen in some of the
patients (SS patients 6 and 8, RA patients 6 and 8, and
SLE patients 3 and 5 , Table 1).
DISCUSSION
In this study, we show that IL-10 gene dysregulation is a common feature of RA, SS, and SLE. This
abnormality was observed in freshly isolated and
1652
LLORENTE ET AL
Table 1. Production of IL-10 by PBMC and MNC subpopulations from Sjogren’s syndrome,
rheumatoid arthritis, and systemic Lupus erythematosus patients*
IL-10 production (pg/ml)
Sjogren’s syndrome
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Patient 7
Patient 8
Patient 9
Patient 10
Mean f SEM
Rheumatoid arthritis
Patient I
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Patient 7
Patient 8
Patient 9
Patient 10
Mean f SEM
Systemic lupus
erythematosus
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Patient 7
Patient 8
Patient 9
Patient 10
Mean ? SEM
~~
~~~~~~
PBMC
T cells
12,895
3,957
12,155
4,735
7,298
4,424
1,035
1,711
2,404
7,907
5,852 1,311
*
9
53
5,037
48
262
0
8
60
0
ND
608 f 554
78
1,337
4,099
1,609
3,437
1,334
60
922
1,052
ND
1,555 f 459
18,000
ND
ND
ND
6,321
590
1,394
350
2,738
3,594
4,712 2 2,347
0
3,515
6,515
6,763
9,262
10,211
ND
385
2,310
1,163
4,458 1,283
5
58
87
243
155
1,721
257
59
145
ND
303 179
0
155
393
2,256
6,505
8,300
714
179
299
ND
2,089 1,039
75
2,713
4,892
4,449
ND
3,598
894
25
1,959
1,335
2,215 f 604
150
2,900
4,040
5,720
10,920
200
720
8,160
6,720
9,200
4,873 +- 1,230
60
20
70
120
150
80
20
310
50
50
93 f 27
320
540
6,480
380
1,420
140
20
2,440
1,700
50
1,349 rt_ 625
500
7,280
1,500
10,920
1,050
730
100
11,240
9,360
1,600
4,428 -C 1,478
_+
~~~~
B cells
_+
_+
~
~
~~
Monocytes
~
* IL-10 = interleukin-10; PBMC = peripheral blood mononuclear cells; MNC = mononuclear cells;
ND = not done.
unstimulated PBMC from untreated patients, which
indicates that it reflects a spontaneous imbalance of
the cytokine network common to these 3 conditions.
Although IL-10 was originally defined as a
product of Th2 lymphocytes, a subpopulation of T
helper cells, it was subsequently shown that macrophages (33), B lymphocytes (34), keratinocytes (39,
and the placenta (36) also produce this cytokine in
vitro. The respective contribution of these different
cell populations in the in vivo production of 1L-10 has
remained uncharacterized. It is therefore important to
point out that in the 3 rheumatic autoimmune diseases
we studied, T lymphocytes contributed very little to
IL-10 production, which originated mainly from B
lymphocytes and monocytes. It thus appears t,.at the
term ‘‘Th2” lymphokine initially proposed to qualify
IL-10 is inappropriate.
As mentioned, IL-10 induces in vitro a strong B
lymphocyte proliferation and differentiation (24),
From these properties, it is likely that the IL-10 gene
dysregulation evidenced in RA, SS, and SLE plays a
major role in the polyclonal B lymphocyte hyperactivity associated with these diseases. Recently, the role
of IL-10 in the pathogenesis of SLE has been suggested from murine models. Administration of neutralizing anti-IL- 10 antibodies delayed the emergence of
autoimmune manifestations, whereas administration
of IL-10 accelerated the onset of the disease (37).
IL-10 IN RHEUMATIC AUTOIMMUNE DISEASES
Whether the role of IL-10 in B lymphocyte-mediated
autoimmunity also applies to humans with SLE, RA,
or SS remains to be determined.
In addition to stimulating B lymphocytes, IL-I0
modulates other immune functions and, in particular,
it deactivates macrophages, inhibiting their production
of cytokines and their ability to stimulate T helper
lymphocytes (38,39). IL-10 also decreases antigen
presentation by dendritic cells (40). The previously
reported impairment of cellular immune responses,
attributed to a defect of accessory cells in SLE (41,42)
may thus be related to the increased production of
IL-10 we found. This may also apply to the decreased
production of TNFa and of IL-6 by stimulated macrophages from SLE patients (18-20,43). This raises the
possibility that part of the effect of IL-10 on rheumatic
autoimmune diseases is to down-regulate immune activity apart from the B lymphocyte response and to
inhibit the inflammatory reaction. The overall effect of
IL-10 hyperproduction on the course of RA, SS, and
SLE might not be identical, depending on the respective contributions in the pathogenesis of B lymphocyte
abnormalities and of inflammatory tissue damage. It
will be important to accurately delineate this role in
each of these autoimmune rheumatic diseases, since
IL-10 might be of therapeutic benefit in some conditions but harmful in others.
The mechanism leading to increased IL-10 production by PBMC in RA, SS, and SLE remains to be
defined. That the level of IL-10 production does not
correlate with any marker of disease activity suggests
that IL-I0 gene dysregulation does not simply reflect
activation of immune cells. This is also supported by
the finding that in patients with HIV infection, another
condition characterized by an overall immune activation and a B lymphocyte hyperactivity (44,45), IL-10
production is significantly lower than in those with
rheumatic autoimmune diseases. Moreover, B lymphocytes in HIV infection do not significantly contribute
to IL-10 production, which arises from both monocytes and CD4- T lymphocytes (Fior R et al: unpublished observations). Our present result is actually the
first evidence that nonmalignant B lymphocytes produce IL-10 in vivo in humans. This suggests that
activated B lymphocytes in RA, SS, and SLE belong
to a subpopulation prone to IL-10 production. This
hypothesis is consistent with the findings of murine
studies, which have shown that B cellderived IL-10
arises mainly from CD5+ B lymphocytes, a subpopulation with an autoreactive repertoire (46).
Although only speculations can be raised at this
1653
time, the relationships between viruses and IL-10
dy sregulation certainly deserves attention in this context. EBV has been shown to be a potent inducer of
IL-I0 production by B lymphocytes (34). Retroviruses
may act in synergy with EBV in triggering IL-10
production, as shown in autoimmune deficiency syndrome lymphomas (26-28). Both EBV and retroviruses are thought to play a role in the pathogenesis of
RA, SS, and SLE (4-7). Our present results provide a
potential explanation, linking such infectious agents to
the B lymphocyte abnormalities observed during these
rheumatic autoimmune diseases.
In addition to IL-10, other cytokines certainly
contribute to B lymphocyte hyperactivity in these
disorders. As mentioned, increased levels of IL-6 are
produced in RA and in SLE (9,18), and this contributes to B lymphocyte hyperactivity in both disorders
(refs. 21 and 22, and Fior et al: unpublished results).
IL-I0 decreases the production of IL-6 by monocyte/
macrophages (33), but not by B lymphocytes and
endothelial cells (47). Actually, IL-10 may activate
endothelial cells in vivo (48). These contrasting effects
of IL-10 on monocytes versus other cell populations
may explain why, in SLE, monocytes produce low
amounts of IL-6 upon stimulation, whereas IL-6 serum
levels are increased and B lymphocytes produce IL-6.
TNFa is another cytokine of interest in the
understanding of B lymphocyte abnormalities in rheumatic autoimmune diseases. As already mentioned,
treatment of RA patients with anti-TNFa antibodies
decreases circulating levels of rheumatoid factors and
improves the clinical features of the disease (17). In
SLE, the production of immunoglobulins by cultured
PBMC is decreased by antibodies to TNF (43). It has
been suggested that the enhancing effect of TNFa on
immunoglobulin production is mediated by an induction of IL-6 production (22). These observations and
our present report indicate that B cell-mediated autoimmunity presumably involves several cytokines acting in concert, and that a combination of several
antagonistic agents may be required to fully inhibit
autoantibody production.
With respect to IL-10 in RA, SS, and SLE,
inasmuch as dysregulation of its gene occurs in both B
lymphocytes and monocytes, its role in the inappropriate stimulation of B lymphocytes would result from
an autocrine as well as a paracrine pathway. Analysis
of the mechanism of IL-10 gene dysregulation and of
the effects of IL-10 in RA, SS, and SLE will be an
important step in our understanding of the pathogenesis of rheumatic autoimmune diseases. This may, in
LLORENTE ET AL
1654
turn, allow investigations of drugs modulating IL- 10
production and effects in these conditions.
REFERENCES
1. Nepom GT, Erlich H: MHC class-I1 molecules and autoimmunity. Annu Rev Immunol9493-525, 1991
2. Siege1 M, Lee SL, Peress NS: The epidemiology of druginduced systemic lupus erythematosus. Arthritis Rheum 10:
407415, 1967
3. Ansar-Ahmed S, Penhale WJ, Talal N: Sex hormones, immune
responses, and autoimmune disease. Am J Pathol 12531-551,
1985
4. Kreig AM, Steinberg AD: Retroviruses and autoimmunity. J
Autoimmun 3:137-166, 1990
5 . Venables P, Brooks S: Retroviruses: potential aetiological
agents in autoimmune rheumatic disease. Br J Rheumatol 31:
841-846, 1992
6. Ngou J, Graafland H, Segondy M: Antibodies against polypeptides of purified Epstein-Ban: virus sera of patients with connective tissue diseases. J Autoimmun 5:243-249, 1992
7. Garzelli C, Paccardi F, Basolo F, Falcone G: Mechanism other
than polyclonal B cell activation possibly involved in EpsteinBarr virus-induced autoimmunity. Clin Exp Immunol 76412416, 1989
8. Noun AME, Panayi GS, Goodman SM: Cytokines and the
chronic inflammation of rheumatic disease. I. The presence of
interleukin-1 in synovial fluids. Clin Exp Immunol 59295-302,
1984
9. Sack U, Kinne R, Marx T, Bender S , Emmrich F: Interleukin-6
in synovial fluid is closely associated with chronic synovitis in
rheumatoid arthritis. Rheumatol Int 13:45-51, 1993
10. Arend W: Interleukins and arthritis: IL-1 antagonism in inflammatory arthritis. Lancet 341:155-156, 1993
11. Saxne T, Palladino MA Jr, Heineiard D, Talal N, Wollheim FA:
Detection of tumor necrosis factor a but not tumor necrosis
factor p in rheumatoid arthritis synovial fluid and serum.
Arthritis Rheum 31:1041-1045, 1988
12. Saklatvala J, Sarsfield SJ, Townsend Y: Pig interleukin-1:
purification of two immunologically different leukocyte proteins
that cause cartilage resoption, lymphocyte activation and fever.
J Exp Med 162:1208-1222, 1985
13. Thomas BM, Mundy GR, Chambers TJ: Tumor necrosis factor
LY and p induce osteoblastic cells to stimulate osteoclast bone
resorption. J Immunol 138:775-779, 1987
14. Henderson B, Pettipher E R Arthritogenic actions of recombinant IL-1 and tumor necrosis alpha in the rabbit: evidence for
synergistic interactions between cytokines in vivo. Clin Exp
Immunol75:306-310, 1988
15. Butler DM, Vitti GF, Leizer T, Hamilton JA: Stimulation of the
hyaluronic acid levels of human synovial fibroblasts by recombinant human tumor necrosis factor a,tumor necrosis factor p
(lymphotoxin), interleukin-1a, and interleukin-lp. Arthritis
Rheum 31:1281-1289, 1988
16. Campbell IK, Piccoli DS, Roberts MJ, Muirden KD, Hamilton
JA: Effects of tumor necrosis factor a and p on resorption of
human articular cartilage and production of plasminogen activator by human articular chondrocytes. Arthritis Rheum 33:
542-552, 1990
17. Elliott MJ, Maini RN, Feldmann M, Long-Fox A, Charles P,
Katsikis P, Brennan FM, Walker J, Bijl H, Ghrayeb J, Woody
JN: Treatment of rheumatoid arthritis with chimeric monoclonal
antibodies,to tumor necrosis factor a. Arthritis Rheum 36:16811690, 1993
18. Sponk PE, Ter Borg EJ, Limburg PC, Kallenberg CGM: Plasma
concentration of interleukin-6 in systemic lupus erythematosus:
an indicator of disease activity? Clin Exp Immunol90:10~110,
1992
19. Linker-Israeli M, Deans R Dysregulated lymphokine production in systemic lupus erythemaiosus. Ann-N Y Acad Sci
5571567-569, 1989
20. Kitani A, Hara M, Hirose T, Harigai K, Suzuki M, Kawakami
Y, Kawaguchi T, Hidaka M, Kawagoe M, Nakamura H: Autostimulatory effects of IL-6 on excessive B cell differentiation in
patients with systemic lupus erythematosus: analysis of interleukin-6 production and IL-6 expression. Clin Exp Immunol
88:75-83, 1992
21. Nafaguchi H, Suzuki N, Mizushima Y, Sakane T: Constitutive
expression of IL-6 receptors and their role in the excessive B
cell function in patients with systemic lupus erythematosus. J
Immunol 151:65256534, 1993
22. Linker-Israeli M, Deans RJ, Wallace DJ, Prehn J, Ozeri-Chen
T, Klinenberg JR: Elevated levels of endogeneous IL-6 in
systemic lupus erythematosus: a putative role in pathogenesis. J
Immunol 147:117-123, 1991
23. Maury CPJ, Teppo A-M: Tumor necrosis factor in the serum of
patients with systemic lupus erythematosus. Arthritis Rheum
32:146-150, 1989
24. Rousset F, Garcia E, Defrance T, Peronne C, Vezzio D, Hsu R,
Kastelein R, Moore KW, Banchereau J: Human and viral IL-10
are potent growth and differentiation factors for activated human B lymphocytes. Proc Natl Acad Sci U S A 89:189&1893,
1991
25. Levy Y, Brouet J-C: Interleukin-10prevents spontaneous death
of germinal center B cells by induction of the bcl-2 protein. J
Clin Invest 93:424-428, 1994
26. Benjamin D, Knobloch TJ, Dayton MA: Human B-cell
interleukin-10: B-cell lines derived from patients with acquired
immunodeficiency syndrome and Burkitt’s lymphoma constitutively secrete large quantities of interleukin-10. Blood 5 : 128%
1298, 1992
27. Emilie D, Touitou R, Raphael M, Peuchmaur M, Devergnee 0,
Rea D, Coumbraras J, Crevon M-C,Edelman L, Joab I,
Galanaud P: In vivo production of interleukin-10 by malignant
cells in AIDS lymphomas. Eur J Immunol22:2937-2942, 1992
28. Emilie D, Galanaud P, Raphael M, Joab I: Interleukin-10 and
acquired immunodeficiency syndrome lymphomas. Blood 81:
1106,1992
29. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield
NF, Schaller JG, Talal N, Winchester RJ: The 1982 revised
criteria for the classification of systemic lupus erythematosus.
Arthritis Rheum 25:1271-1277, 1982
30. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF,
Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS,
Medsger TA Jr, Mitchell DM, Neustadt DH, Pinals RS, Schaller
JG, Sharp JT, Wilder RL, Hunder GG: The American Rheumatism Association 1987 revised criteria for the classification of
rheumatoid arthritis. Arthritis Rheum 31:315-324, 1988
31. Fox RI, Robinson CA, Curd JG, Kozin F, Howell FV: Sjogren’s
syndrome: proposed criteria for classification. Arthritis Rheum
29577-585, 1986
32. G uz m h J, Cardiel MH, Arce-Salinas A, SBnchez-GuerreroJ,
Alarc6n-Segovia D: Measurement of disease activity in systemic lupus erythematosus: prospective validation of 3 clinical
indices. J Rheumatol 19:1551-1558, 1992
33. De Waal Malefyt R, Abrams J, Bennett B, Figdor C, de Vries J:
Interleukin 10 (IL-10) inhibits cytokine synthesis by human
monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 174:1209-1220, 1991
34. Burdin N, Pkronne C, Banchereau J, Rousset F: Epstein-Barr
virus transformation induces B lymphocytes to produce human
interleukin 10. J Exp Med 177:295-304, 1993
IL-10 IN RHEUMATIC AUTOIMMUNE DISEASES
35. Enk AH, Katz SI: Identification and induction of keratinocytederived IL-10. J Immunol 149:92-95, 1992
36. Lin H, Mosmann TR, Guilbert L, Tuntipopipat S, Wegmann
TG: Synthesis of T helper 2-type cytokines at the maternal-fetal
interface. J Immunol 151:4562-4573, 1993
37. Ishida H, Muchamuel T, Sakaguchi S, Andrade S, Menon S,
Howard M: Continuous administration of anti-interleukin-10
antibodies delays onset of autoimmunity in NZB/WFl mice. J
Exp Med 179:305-310, 1994
38. Fiorentino JF, Zlotnik A, Mosmann TR, Howard M, O’Garra
A: IL-10 inhibits cytokine production by activated macrophages. J Immunol 147:3815-3822, 1991
39. D’Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M,
Trinchieri G: Interleukin 10 (IL-10) inhibits human lymphocyte
interferon yproduction by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med
178~1041-1048,1993
40. Enk AH, Angeloni VL, Udey MC, Katz SI: Inhibition of
Langerhans cell antigen-presenting function by IL-10: a role for
IL-10 in induction of tolerance. J Immunol 151:2390-2398, 1993
41. Crow MK: Enhancement of the impaired autologous mixed
leukocyte reaction in patients with systemic lupus erythematosus. J Clin Invest 76:807-815, 1985
42. Via CS, Tsokos GC, Bermas B, Clerici M, Shearer GM: T
cell-antigen-presenting cell interactions in human systemic lu-
1655
pus erythematosus: evidence for heterogeneous expression of
multiple defects. J Immunol 151:3914-3922, 1993
43. MalavC I, Searles RP, Montano J, Williams RC: Production of
tumor necrosis factor/cachectin by peripheral blood mononuclear cells in patients with systemic lupus erythematosus. Int
Arch Allergy Appl Immunol89:355-361, 1989
44. Emilie D, Peuchmaur M, Maillot MC, Crevon MC, Brousse N,
Delfraissy JF, Dormont J, Galanaud P: Production of interleukins in human immunodeficiency virus-1-replicating lymph
nodes. J Clin Invest 86:148-159, 1990
45. Lane HC, Masur H, Edgar LC, Whalen G, Rook AH, Fauci AS:
Abnormalities of B-cell activation and immunoregulation in
patients with the acquired immunodeficiency syndrome. N Engl
J Med 309:453-458, 1983
46. O’Garra A, Chang R, Go N, Hastings R, Haughton G, Howard
M: Ly-1 B (B-1) cells are the main source of B cell-derived
interleukin-10. Eur J Immunol22:711-717, 1992
47. Sironi M, Munoz C, Pollicino T, Siboni A, Sciacca FL, Nernasconi S, Vecchi A, Colotta F, Mantovani A: Divergent effects of
interleukin-10 on cytokine production by mononuclear phagocytes and endothelial cells. Eur J Immunol23:2692-2695, 1992
48. Wogensen L, Huang X,Sarvetnick N: Leukocyte extravasation
into the pancreatic tissue in transgenic mice expressing
interleukin-10 in the islets of Langerhans. J Exp Med 178:175185, 1993
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