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The presence of interleukin-13 in rheumatoid synovium and its antiinflammatory effects on synovial fluid macrophages from patients with rheumatoid arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 39, No. 10, October 1996, pp 1693-1702
8 1996, American College of Rheumatology
1693
THE PRESENCE OF INTERLEUKIN-13 IN RHEUMATOID SYNOVIUM
AND ITS ANTIINFLAMMATORY EFFECTS ON SYNOVIAL FLUID
MACROPHAGES FROM PATIENTS WITH RHEUMATOID ARTHRITIS
PIA ISOMAKI, REIJO LUUKKAINEN, PAAVO TOIVANEN, and JUHA PUNNONEN
Objective. To study the production of
interleukin-13 (IL13) in rheumatoid synovium and the
effects of recombinant IL-13 on the phenotype and
function of synovial fluid (SF) macrophages and T cells
derived from patients with rheumatoid arthritis (RA).
Methods. The presence of IL-13 in SF was studied
using an IL- 13-specific enzyme-linked immunosorbent
assay (ELISA); the production of IL-13 was studied in
SF mononuclear cells (SFMC) by reverse transcriptasepolymerase chain reaction. The effects of recombinant
IL-13 on cytokine production by and phenotype of
SFMC were evaluated using cytokine-specific ELISAs
and flow cytometry, respectively. The effect of IL-13 on
the proliferation of SFMC was determined by 3Hthymidine incorporation. The production and the effects
of IL-13 were compared with those of IL4.
Results. IL-13 was present in 27 of 28 SF samples,
and IG13 messenger RNA (mRNA) was detectable in
SFMC. Importantly, IL-13 levels were significantly
higher than those of IL-4, and IL-13 protein and mRNA
were Fxpressed in several samples, although IL-4 synthesis was undetectable. Recombinant IL-13 significantly reduced the production of IL-lP and tumor
necrosis factor (Y and the expression of CD16 and CD64
by SF macrophages, whereas the expression of HLA-DR
and CD23 was increased. These effects on SF macroPresented in part at the Sixteenth European Workshop for
Rheumatology Research, Stockholm, Sweden, March 14-16, 1996.
Supported in part by grants from the Academy of Finland and
Leiras Research Foundation. DNAX Research Institute is supported
by the Schering-Plough Corporation.
Pia Isomaki, MD, Paavo Toivanen, MD: Turku Immunology
Centre, and Turku University, Turku, Finland; Reijo Luukkainen,
MD: Satalinna Hospital, Harjavalta, Finland; Juha Punnonen, MD:
Turku University, Turku, Finland, and DNAX Research Institute,
Palo Alto, California.
Address reprint requests to Pia Isomaki, MI), Department of
Medical Microbiology, Turku University, Kiinamyllynkatu 13, FIN20520 Turku, Finland.
Submitted for publication January 22, 1996; accepted in
revised form May 6, 1996.
phages were similar to those observed with IL-4, but in
contrast to IL-4, IL-13 had no growth-promoting effect
on SF T cells.
Conclusion. IG13 is consistently present in rheumatoid synovium. The ability of exogenous IL-13 to
decrease the production of proinflammatory cytokines
by SFMC suggests that it may have therapeutic potential in the treatment of patients with RA.
Rheumatoid arthritis (RA) is an autoimmune
disease characterized by accumulation of T cells, macrophages, and plasma cells in the joints. Macrophagederived cytokines, such as interleukin-1 (IL-l), IL-6,
IL-10, tumor necrosis factor CY (TNFcu), and granulocytemacrophage colony-stimulating factor (GM-CSF), are
abundant in rheumatoid synovium (1-6). Cytokines produced by T cells, such as IL-2, IL-4, and interferon-y
(IFNy), have also been demonstrated in rheumatoid
joints, although the levels of expression of these cytokines appear to be lower than those produced by macrophages (7-11). Despite the relatively low concentrations
of T cell-derived cytokines in the joints of patients with
RA, the phenotype of synovial T cells shows that they
are activated (12,13), and it has been suggested that T
cells are important in the induction and maintenance of
synovial inflammation in patients with RA (14,15). Recently, synovial T cells derived from patients with RA
were shown to cause inflammatory arthritis when transferred into severe combined immunodeficient mice (16),
further supporting the notion that T cells and T cellderived cytokines play a major role in the disease
process that occurs in rheumatoid synovium.
The outcome of several infectious and inflammatory diseases has been suggested to depend on the
cytokine production profile of specific T helper cells,
based on which human and mouse CD4+ cells are
divide,dinto 3 major subsets (17). Thl cells produce IL-2
and IFNy, whereas Th2 cells generally secrete high
1694
levels of IL-4, IL-5, and IL-13. Tho cells produce both
Thl and Th2 cytokines. Thl cytokines are involved in
delayed hypersensitivity reactions and promote activation of inflammatory cells such as monocyte/macrophages, whereas Th2 cytokines help with antibody production. In RA, there is evidence of preferential Thl
activation in the synovium, although some synovial T cell
clones also produce significant amounts of IL-4
(9,18,19). Studies using animal models suggest that Thl
cytokines promote the development of autoimmune
disorders, such as experimental autoimmune encephalomyelitis and insulin-dependent diabetes mellitus,
whereas Th2 cytokines may attenuate these diseases (20).
I L 4 is a Th2 cytokine known to have pleiotropic
effects on the regulation of immune responses. IL-4
expresses B cell-, T cell-, and mast cell-stimulating
activities(21,22)but suppresses the function of monocyte/
macrophages by decreasing the cytotoxic activity and the
production of cytokines (IL-1, IL-6, IL-8, IL-12, TNFa,
GM-CSF, and G-CSF) by these cells (23,24). IL-4 has
been suggested to attenuate the inflammatory process in
rheumatoid synovium, because it inhibits the production
of proinflammatory cytokines and immunoglobulins by
pieces of RA synovium (25) and decreases bone resorption and synoviocyte proliferation in vitro (26,27). In
addition, systemically injected IL-4 has been shown to
suppress the chronic destructive phase of streptococcal
cell wall-induced arthritis in rats (28).
Human IL-13 was recently cloned and sequenced
(29,30), and was subsequently found to share many, but
not all, functional properties with IL-4. IL-13 is -25%
homologous to IL-4, and the gene encoding IL-13 is
located on chromosome 5q31 in close proximity to that
encoding IL-4, which suggests that the 2 genes are the
result of gene duplication (31). Like IL-4, IL-13 downregulates the production of proinflammatory cytokines
and suppresses the cytotoxic function of monocyte/
macrophages (24). It also induces B cell proliferation
and IgG4 and IgE synthesis by normal and immature
human B cells (32,33). In addition, IL-13 has been
shown to suppress experimental autoimmune encephalomyelitis in rats (34), demonstrating the antiinflammatory effects of IL-13 in vivo.
In the present study, we demonstrate that IL-13 is
consistently present in synovial fluid (SF) samples derived from patients with RA. Recombinant IL-13 significantly reduced the production of IL-lP and TNFa by
SF mononuclear cells (SFMC), indicating that it has
antiinflammatory effects on SF macrophages. The effects of IL-13 on SF macrophages were similar to those
of IL-4, but IL-13 lacked the T cell growth-promoting
ISOM-
ET AL
activity of IL-4. These data indicate that IL-13 is produced in rheumatoid synovium and suggest that it may
have therapeutic potential in the treatment of RA.
PATIENTS AND METHODS
Patients. Forty-seven patients with RA (35 women and
12 men) were enrolled in the study. The median age of the
patients was 61 years (range 2F80), and the median duration
of disease was 18 years (range 0.5-39 years). The mean (? SD)
erythrocyte sedimentation rate (ESR) was 59 ? 28 mm/hour
and the C-reactive protein (CRP) level was 44 -t 27 mg/liter at
the time the samples were taken. RA was determined according to the criteria of the American College of Rheumatology
(formerly, the American Rheumatism Association) (35).
Eleven patients had seronegative RA. Twenty-nine patients
were receiving disease-modifying antirheumatic drugs
(DMARDs), 28 patients were receiving steroids, and 42 patients were taking nonsteroidal antiinflammatory drugs. This
study was approved by the ethics committee of Turku
University.
Reagents. Purified recombinant human IL-4 (rHuIL-4;
specific activity 1 X lo7 units/mg), rHuIL-10 (2 X lo7 units/
mg), and rHuIL-13 (5 X lo6 units/mg), and neutralizing
anti-IL-4 (25D2) (32), anti-IL-10 (19F1) (36), and anti-IL-13
(2F9) MAb were kindly provided by Dr. Jan E. de Vries
(DNAX Research Institute, Palo Alto, CA). Fluorescein isothiocyanate (F1TC)-and phycoerythrin (PE)-conjugated antihuman CD3, CD14, CD16, CD23, CD25, CD69, HLA-DR,
and mouse IgG with irrelevant specificity were purchased from
Becton Dickinson (Mountain View, CA). FITC-conjugated
anti-human CD64 was purchased from Pharmingen (San
Diego, CA).
Cell preparations and culture conditions. SF samples
from the inflamed knee joints of RA patients were obtained by
needle aspiration into heparinized tubes. After centrifugation,
SF supernatants were stored at -70°C until examined for the
presence of cytokines by enzyme-linked immunosorbent assay
(ELISA). For comparison, blood samples were collected from
healthy donors.
SFMC or peripheral blood mononuclear cells (PBMC)
were isolated by a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density-gradient centrifugation and washed twice with
phosphate buffered saline (PBS) at 4°C. Macrophages were
enriched from cell suspensions by adherence to plastic petri
dishes (Bibby Sterilin Ltd, Stone, England) for 1 hour at 37°C.
This adherent cell population, referred to as purified macrophages, contained >80% macrophages, as judged by antiCD14 staining and flow cytometry.
Purified macrophages, nonadherent cells, or total mononuclear cells were cultured at 106/mVwell, or for studies of
cytokine production, at 2 X 1O6/m1/well, in Iscove’s modified
Dulbecco’s medium (Gibco BRL, Paisley, Scotland) supplemented with 10% heat-inactivated fetal calf serum (FCS;
Bioproducts for Science, Indianapolis, IN) and 50 pg/ml
gentamicin (Biological Industries, Bethaemek, Israel). Cells
were cultured in 24-well plates (Costar, Cambridge, MA) at
37°C in a humidified atmosphere containing 5% CO,. IL-4,
IL10, IL-13, or neutralizing monoclonal antibody (MAb) at
concentrations indicated herein was added at the beginning of
IL-13 IN RHEUMATOID ARTHRITIS
the cultures. To study cytokine production, lipopolysaccharide
(LPS) (1 pglml, from Eschen’chia coli 0127:B8; Sigma, St.
Louis, MO) was added to the cultures together with the
cytokines, and supernatants were collected after 24 hours.
Supernatants were centrifuged briefly to remove cell debris
and stored at -70°C until used for ELISA.
Flow cytometry. Cells were harvested after a culture
period of 3 days and washed twice with PBS supplemented with
2% FCS containing 0.01% sodium azide. Cells were then
incubated with 1% normal mouse serum (Dako, Glostrup,
Denmark) for 30 minutes at 4°C. After 2 washes, FITC- and
PE-conjugated MAb were added at saturating concentrations
for 30 minutes at 4°C. An isotype-matched mouse IgG with
irrelevant specificity was used as a negative control antibody.
Two additional washes were performed, and labeled cells were
analyzed using a FACScan (Becton Dickinson) flow cytometer
and Lysys I1 software. To quantify the effects of IL-4 and IL13
on the expression of surface antigens, the mean fluorescence
intensity for each staining was measured.
Measurement of cytokine levels. Cytokine levels in the
SF samples and culture supernatants were determined using
cytokine-specific ELISAs, which were either described previously or obtained commercially and used according to the
manufacturers’ instructions. IL-4 (37), IL-13 (38), and TNFa
(39) levels were measured as described. The sensitivity of the
IL-4 and TNFa ELISAs was 50 pglml; that of the IL-13 ELISA
was 150 pglml. IL-1p levels were measured using ELISA kits
obtained from Cistron Biotechnology (Pine Brook, NJ). The
sensitivity of this ELISA was 30 pglml. All ELISAs were
performed in duplicate.
To eliminate the possible influence of rheumatoid
factors (RF) in the IL-4 and IL-13 ELISAs, all SF samples
were incubated for 1 hour at 4°C with f i e d Staphylococcus
aureus cells that had high surface levels of protein A (Pansorbin cells; Calbiochem, La Jolla, CA). Thereafter, the samples were centrifuged at 10,OOO revolutions per minute for 5
minutes and supernatants were collected. This treatment was
performed twice and removed >80% of the total Ig from the
SF samples, as measured by ELISA, but did not significantly
affect the levels of IL-4 or IL-13. The levels of IL-4 detected
before and after the treatment were 240 ? 98 pg/ml (mean +SEM) and 173 ? 38 pg/ml, respectively, and those of IL-13
were 1,081 ? 257 pglml and 1,307 -t 247 pg/ml, respectively.
Proliferation assays. Cells (lo’) were cultured in triplicate in 200 p1 of RPMI supplemented with 10% heatinactivated normal human serum, 0.1 mM mercaptoethanol
(Sigma), and 50 pg/ml of gentamicin in flat-bottom 96-well
microtiter plates (Costar) for 4 days. IL-4 or IL-13 was added
at the start of culture, at the concentrations indicated herein.
3H-thymidine (1.0 pCi/well) was added for the final 18 hours of
culture, and the cells were harvested and analyzed by scintillating counting (Wallac, Turku, Finland).
Messenger RNA (mRNA) isolation and reverse
transcriptase-polymerase chain reaction (RT-PCR)analysis.
Total RNA was extracted from freshly isolated SFMC, as
described by Chirgwin et a1 (40). Briefly, cells were homogenized in 4M guanidinium isothiocyanate, and RNA was purified by ultracentrifugation through a cesium chloride cushion.
Two micrograms of total RNA was reverse transcribed using
Superscript reverse transcriptase (Gibco BRL, Gaithersburg,
MD) and oligo(dT),,,,
as primer (Gibco BRL), according to
1695
the procedure of Krug and Berger (41), in a 20-p1 reaction. An
amount equivalent to 50 ng of total RNA was used for the
amplification reactions. Each 50-pl PCR reaction contained 1
unit of AmpliTuq DNA polymerase (Perkin Elmer, Norwalk,
0,
5 pl of lox PCR buffer (Perkin Elmer), 1 mM MgCI,,
200 ~ L of
M each dNTP, and 25 nmoles of each primer.
The following primers, kindly provided by Dr. Rene de
Waal Malefyt (DNAX Research Institute), were used: IL-4
sense, 5’-ACTGCTTCCCCCTCTGTTTCTTCCTG-3’;
IL-4
antisense, 5’-ACGTACTCTGGTTGGCTTCCTTCAC-3’;
IL13 sense, 5’-CCCAGAACCAGAAGGCTGCG-3‘;
IL-13
antisense, 5‘-CAGTTGAWCGTCCCECCG-3’;hypoxanthineguanine phosphoribosyltransferase (HPRT) sense, 5’-CTGGCGTCGTGATTAGTGATGATGA-3’;and HPRT antisense,
5‘-CITAGGCITTGTATI?TGCZTTTCC-3’.
Reaction mixtures were incubated in a Perkin-ElmerCetus DNA Thermal Cycler 9600 for 40 cycles (denaturation
for 30 seconds at 94“C, annealing for 30 seconds at 55”C, and
extension for 60 seconds at 72°C). Twenty microliters of each
PCR product was electrophoresed through 1.2% agarose and
visualized as ultraviolet fluorescence after staining with
ethidium bromide. Gels were denatured in 3M NaCI, 0.4M
NaOH, and transferred to a Nytran membrane (Schleicher &
Schuell, Keene, NH) according to the manufacturer’s instructions. Membranes were prehybridized in QuickHyb solution
(Stratagene, La Jolla, CA) for 2 hours at 60°C.
Oligonucleotide probes specific for a sequence internal
to the primers used in the amplification were labeled with
y-”P-ATP (Amersham, Arlington Heights, IL) using T4
polynucleotide kinase (Stratagene) for 5’-end labeling as described (42). To remove unincorporated nucleotides, probes
were run through a Nick column (Pharmacia). Following
hybridization for 3 hours at 60°C, membranes were washed in
2X saline sodium citrate-0.1% sodium dodecyl sulfate for 20
minutes at room temperature and for 30 minutes at 55°C.
Membranes were exposed to Kodak X A R - 5 film for 2-24
hours. Oligonucleotide probes were: IL-4, 5’-CAGTTCCACAGGCACAAGCAG-3’ and IL-13, 5’-GCATCGAGAAGACCCAGAGG-3 ‘
Statistical analysis. Statistical analysis was performed
using Wilcoxon signed rank test. Correlations were calculated
using Spearman’s rank correlation method. Limit values were
used for ELISA levels below or above the detection limits of
the ELISAs.
RESULTS
Production of IL-13 in the joints of patients with
RA. We first studied the levels of IL-13 in freshly
isolated SF samples from 28 patients with RA by
cytokine-specific ELISAs. The results were compared
with IL-4 levels in the same samples. To eliminate any
possible influence of RF,all SF samples were incubated
twice with fixed S aureus cells that had high surface
levels of protein A (see Patients and Methods).
IL-13 was detectable in 27 of the 28 samples
(detection limit 150 pg/ml), whereas IL-4 was detectable
(>50 pg/ml) in 18 of the 28 samples (Table 1). The levels
ISOMAKI ET AL
1696
Table 1. Levels of interleukin-4 (IL-4) and IL-13 in synovial fluid
from patients with rheumatoid arthritis*
Cytokine level
(PiiW)
Patient
IL-4
IL-13
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
<50
60 t 2
<50
73 t 6
<50
513 f 22
930 2 38
1,036 -C 6
1,186 f 60
1,059 2 153
658 ? 11
897 2 40
1,119 f 250
1,186 2 12
1,536 i- 50
492 2 61
236 t 69
196 t 0
488 i- 30
<150
259 2 0
>5000
1,105 ? 294
1,032 2 175
>5,000
908 2 15
1,028 2 145
4,410 2 578
1,184 f 127
276 2 43
1,564 2 163
1,630 2 0
1,533 2 0
<50
<50
6 6 ? 11
81 t 2
175 ? 16
<so
<50
192 ? 3
<50
<so
650 2 19
453 f 38
768 i- 61
126 2 16
638 2 179
88 f 0
109 2 2
151 i- 9
189 ? 18
<50
361 f 9
123 2 5
66 2 0
* Cytokine levels were determined by specific enzyme-linked immunosorbent assays as described in Patients and Methods. Values are the
mean f SEM of duplicate determinations.
of IL-13 (mean ? SEM 1,307 -+ 247 pg/ml) were
significantly higher (P < 0.001) than those of IL-4
(173 ? 38 pdml). IL-13 levels >1,000 pdml were
detected in 16 of the 28 patients, whereas the level of
IL-4 was <1,000 pdml in every patient tested. The levels
of IL-4 and IL-13 did not correlate with the ESR, the
CRP level, age, or disease duration (P > 0.05; data not
shown). In addition, no apparent influence of DMARDs
or steroids on the levels of IL-4 and IL-13 was observed
(data not shown).
To investigate whether IL-4 and IL-13 were
produced locally by synovial cells, the presence of IL-4
and IL-13 mRNA in SFMC from 8 patients with RA was
studied by RT-PCR and Southern hybridization. As
shown in Figure 1, IL-13 mRNA could be detected in 7
of the 8 SFMC samples. Interestingly, IL-13 mRNA was
detectable in 3 SFMC samples that were negative for
IL-4 (samples 3,5, and 7), and 1 sample that was positive
for IL-4 was negative for IL-13 (sample 8), indicating
that IL-4 and IL-13 are not coexpressed in every patient.
These data demonstrate that IL-13 is produced in
rheumatoid synovium, and is present more consistently
and at higher levels than IL-4 in SF samples from
patients with RA.
Effects of IL-13 on IL-lP and TNFa production
by SFMC. We next investigated the effects of IL-13 on
the production of the proinflammatory cytokines IL-1p
and T N F a by SFMC from patients with RA, and these
effects were compared with those of IL-4. No spontaneous production of IL-1p and T N F a was detectable in
vitro; therefore, LPS was added to enhance the activation state of the macrophages. SFMC were cultured for
24 hours in the presence of 100 units/ml of IL-4 or IL-13,
which was the optimum dose for suppressing cytokine
production (data not shown).
Both IL-4 and IL-13 significantly inhibited the
production of IL-1p and T N F a by SFMC ( P < 0.05)
(Figure 2), and no significant difference between the
effects of IL-4 and IL-13 was observed. Since we have
previously shown that IL-10 decreases the production of
IL-1p and TNFa by SFMC (4), we compared the effects
of IL-4 and IL-13 with those of IL-10. As shown in
Figure 2, IL-10 was a more potent down-regulator of
1 2 3 4 5 6 7 0
IL-13 mRNA
IL-4 mRNA
HPRT mRNA
Figure 1. Expression of interleukin-4 (IL-4) and IL-13 messenger
RNA (mRNA) by synovial fluid mononuclear cells (SFMC) from 8
patients with rheumatoid arthritis. Total RNA from SFMC was
isolated and subjected to reverse transcription. Complementary DNA
(cDNA) amplified by primers specific for hypoxanthine-guanine phosphoribosyltransferase (HPRT) was detected by ethidium bromide
staining; cDNA specific for IL-4 and IL-13 was analyzed using
Southern blotting (see Patients and Methods).
IL-13 IN RHEUMATOID ARTHRITIS
1697
1200
T
D
T
Figure 2. Effects of interleukin-4 (IL-4) and DL13 on the production of IL-lp and tumor necrosis factor a (TNFa) by lipopolysaccharide
(LPS)-stimulated synovial fluid mononuclear cells (SFMC) from patients with rheumatoid arthritis. SFMC were cultured in the presence. of 100
units/ml of IL-4 or IL-13 (n = 8), 100 units/rnl of IL-10 (n = 5 ) , and/or 5 Fg/ml of neutralizing anti-IL-10 monoclonal antibody ( a L 1 0 ; n = 6).
LPS (1 pg/rnl) was added to all cultures. Supernatants were collected after a culture period of 24 hours, and the production of IL-lp (A and B) and
TNFa (C and D) was determined using cytokine-specific enzyme-linked immunosorbent assays. A mean 2 SEM level of 661 ? 98 p & ' d of IL-lp
and 749 -t 241 pg/ml of TNFa was produced in the presence of LPS alone. Bars show the mean and SEM percentage of control.
cytokine production than was either IL-4 or IL-13.
Importantly, however, the addition of IL-4 or IL-13 to
IL-10-treated cultures further down-regulated the production of IL-1p (P < 0.05 versus IL-10 alone). In
contrast, TNFa production was not altered by I L 4 or
IL-13 in the presence of IL-10.
To study the effects of IL-4 and IL-13 in the
absence of endogenous IL-10, anti-IL-10 MAb were
added to the cultures. As shown in Figure 2, IL-4 and
IL-13 strongly inhibited the production of both IL-1p
and TNFa in the presence of anti-IL-10 MAb ( P < 0.05
versus anti-IL-10 MAb alone). We also investigated
whether endogenously produced IL-4 or IL-13 affects
cytokine production by SFMC. However, no IL-4 or
IL-13 production by SFMC could be observed using
ELISA, regardless of whether LPS was added (n = 12;
data not shown). Consistent with these results, neutralizing anti-IL-4 or anti-IL-13 MAb had no significant
effects on IL-1p or TNFa production by SFMC (n = 4;
data not shown). Nevertheless, our results demonstrate
that exogenous IL-4 and IL-13 down-regulate the production of proinflammatory cytokines by SFMC, and
they function independently of IL-10.
Effects of IL-13 on the phenotype of SF macrophages and T cells. Next, we studied the effects of IL-13
on the expression of HLA-DR, CD16, CD23, and CD64
ISOM&
1698
ET AL
Table 2. Effects of interleukin-4 (IL-4) and IL-13 on the phenotype of CD14+ synovial fluid (SF) macrophages from 7 patients with rheumatoid
arthritis and peripheral blood (PB) monocytes from 8 healthy donors'
SF macrophages
PB monocytes
Antigen
Medium
IL-4
IL-13
Medium
HLA-DR
CD16
CD24
CD64
198 f 49
30 t 9
420
12 t 3
276 5 65
822
14 t 5
621
247 t 67
12 f 3
11 5 4
721
147 2
17 2
4 t
9 t
24
2
0
l
IL-4
IL-13
244 f 31
7 t 1
24 f 3
242 f 45
7 t l
21 t 3
4tO
4fO
* Purified monocyte/macrophages or total mononuclear cells were cultured for 3 days in medium alone or in the presence of IL-4 or I L 1 3 (100
units/ml). Values are the mean f SEM mean fluorescence intensity.
on CD14+ SF cells, and the results were again compared with those observed in response to IL-4. In
preliminary experiments, IL-4 and IL-13 induced maximal phenotypic changes after a culture period of 3 days
(data not shown). Therefore, purified macrophages or
total SFMC from 7 patients with RA were cultured for 3
days in the presence and absence of IL-4 or IL-13 (100
unitdml), and the expression of surface antigens was
analyzed by flow cytometry.
As shown in Table 2, the expression of HLA-DR
and CD23 was increased, whereas the expression of the
Fcy receptors CD16 and CD64 was decreased by IL-4
and IL13, as determined by a change in mean fluores-
HLA-DR
CD16
cence intensity. Figure 3 illustrates the flow cytometry
histograms for a representative patient. No additional
effects were observed when cells were cultured in the
presence of both IL-4 and IL-13 (Figure 3). Neutralizing
anti-IL-4 and anti-IL-13 MAb completely blocked the
effects of IL-4 and IL-13, respectively, illustrating the
specificity of these effects (data not shown). Comparable
results were obtained with purified macrophages and
total SFMC (data not shown).
The effects of IL-4 and IL-13 on SF macrophages
were also compared with the effects on PB monocytes
from healthy volunteers. As shown in Table 2, the effects
of IL-4 and IL-13 on these 2 cell populations were
CD23
CD64
IL-4
IL-13
IL-4
+
IL-13
Figure 3. Effects of interleukin-4 (IL4) and IL-13 on the expression of HLA-DR, CD16, CD23, and CD64 on synovial fluid (SF) macrophages from
a patient with rheumatoid arthritis. Purified SF macrophages were cultured in medium alone or in the presence (shaded areas) or absence (open
areas) of IL-4, IL-13, or I L 4 plus IL-13 (100 units/ml) for 3 days, and the expression of surface antigens on gated CD14+ cells was analyzed by flow
cytometry.
1699
IL-13 IN RHEUMATOID ARTHRITIS
expression of these surface antigens on SF CD3+ cells
was studied by flow cytometry. Neither IL-4 nor IL-13
are consistent with the observations using normal human
T cells (data not shown).
Effect of IG13 on the proliferation of SFMC. In
contrast to IL-4, which has well-established growthpromoting effects on T cells (454,IL-13 did not directly
induce proliferation of normal human PB T cells or T
cell clones (31,46). Recently, however, IL-13 was shown
to induce phosphorylation of the IL-4-dependent transcription factor NF-IL-4 in highly purified human T
cells, suggesting that a functional IL-13 receptor complex is expressed in these cells (47).
To investigate the effects of IL-4 and IL-13 on SF
T cells, proliferation of SFMC in the presence and
absence of these cytokines was studied. As shown in
Figure 4, IL-4 induced the proliferation of SFMC from
patients with RA in a dose-dependent manner. The
increased proliferation was observed at a concentration
as low as 10 units/ml, whereas maximal effects were seen
at 100 units/ml (Figure 4). In contrast, IL-13 had no
significant effect on the proliferation of these cells, even
at a concentration of 1,000 unitdm1 (Figure 4). In
addition, IL-13 had no effect on IL-4-induced proliferation of SFMC (data not shown). The results were
similar regardless of whether unfractionated SFMC or
nonadherent, T cell-enriched cell populations were
studied (data not shown).
DISCUSSION
Our results indicate that IL-13 is consistently
present in the joints of patients with RA. IL-13 was
present at higher levels than IL-4, and in several samples, IL-13 protein and mRNA were expressed but IL-4
synthesis was undetectable. Importantly, exogenous
IL-13 had potent inhibitory effects on cytokine production by SF macrophages, suggesting that endogenous
Cytokine concentration (Ulml)
Figure 4. Effects of interleukin-4 (IL-4) and IL-13 on the proliferation
of synovial fluid mononuclear cells from 6 patients with rheumatoid
arthritis. Cells were cultured in the presence of increasing concentrations of IL-4 or IL-13 for 4 days, and proliferation was determined by
3H-thymidine incorporation during the final 18 hours of culture. Bars
show the mean ? SEM percentage of control from experiments, each
performed in triplicate.
IL-13 production is insufficient for optimum inhibition
of proinflammatory cytokine production in rheumatoid
synovium. The effects of IL-13 on the cytokine production by and the phenotype of SF macrophages were
similar to those of IL-4, but, in contrast to IL-4, IL-13
had no growth-promoting effects on SF T cells. These
data indicate that IL-13 has IL-4-like effects on SF
macrophages, but not on T cells, and suggest that IL-4
and IL-13 have distinct roles in the regulation of synovial
inflammation.
In rheumatoid synovium, IL-13 protein and
mRNA were present more consistently and at higher
levels than IL-4. Earlier studies regarding IL-4 production in the joints of patients with RA have yielded
somewhat conflicting results. It has been reported that
IL-4 is absent or is present in minimal amounts in SF
samples from patients with RA (10). However, our
results are in line with those of studies demonstrating
the production of significant quantities of I L 4 by cultured synovial membranes (ll), as well as the expression
of IL-4 mRNA in SF T cells and synovial membranes
from some, but not all, patients with RA (8,9). In
addition, our results are consistent with those of a recent
study indicating that after T cell activation in vitro, IL-13
is produced for significantly longer periods of time than
IL-4 (38). In the same study, it was demonstrated that
1700
while IL-4 is predominantly a Th2 cytokine, IL-13 is also
produced by Tho- and Thl-type T cell clones following
antigen-specific or polyclonal activation. In addition,
some Th2 clones that produced high levels of IL-13
produced low levels of IL-4 (38). These results may
explain our demonstration of high IL-13 levels compared with the levels of IL-4 and our inability to detect
IL-4 in several samples. Although IL-13 was readily
detectable in SF, no spontaneous production of IL-13 by
SFMC was observed in vitro, which is consistent with the
fact that IL-13 production cannot be detected even by
ThZtype cells unless the cells are activated by antigen or
polyclonal T cell mitogens (38). Therefore, in vivo
studies are necessary to investigate the physiologic significance of IL-13 in patients with RA. Nevertheless, the
fact that IL-13 was detected at higher levels than IL-4
suggests that IL-13 may have a more important role than
IL-4 in the regulation of inflammatory responses in
rheumatoid synovium.
Exogenous IL-13 significantly decreased the production of the proinflammatory cytokines IL-1p and
TNFa by LPS-stimulated SF macrophages. Both IL-1p
and TNFa are able to stimulate collagenase and prostaglandin E production and induce cartilage destruction in
vitro (48-50), and they are arthritogenic in animal
models (51,52). In addition, treatment of RA patients
with anti-TNFa MAb has resulted in clinical improvement (53). Based on these earlier results, IL-1 and
TNFa are thought to play an important role in the
pathogenesis of rheumatoid synovitis; therefore, the
inflammatory process in the synovium may be suppressed by the ability of IL-4 and IL-13 to down-regulate
the production of these cytokines.
Our results concerning the effects of IL-4 and
IL-13 on cytokine synthesis differ somewhat from those
previously reported by Hart et a1 (43,44), who demonstrated that these cytokines decreased the production of
IL-lp, but not TNFa, by LPS-stimulated SFMC from
patients with inflammatory arthritis. Interestingly, we
also found that in the presence of exogenous IL-10, IL-4
and IL-13 decreased only IL-lp, but not TNFa, production, which suggests that the difference in the results
obtained by us and by Hart et a1 may be due to different
levels of endogenous IL-10 induced by LPS activation.
Our demonstration that IL-4 or IL-13 together with
IL-10 suppress IL-lp production in an additive manner
suggests that it might be beneficial to combine either
IL-4 or IL-13 with IL-10 to maximally inhibit the production of proinflammatory cytokines in the joints of
patients with RA.
IL-13 increased the expression of HLA-DR and
ISOMAKI ET AL
CD23 and decreased the expression of the Fcy receptors
CD16 and CD64 by SF macrophages. These effects were
similar to those induced by IL-4, thus supporting the
conclusion that the effects of these cytokines on S F
macrophages are similar. It is not known how this
modulation of macrophage phenotype by IL-13 affects
the inflammatory responses in the synovium. However, since HLA-DR molecules are involved in antigen presentation and T cell activation, the enhanced
expression of HLA-DR may potentiate the inflammatory cascade that occurs in inflamed joints. The
decrease in Fcy receptor expression may in turn
reduce the activation of synovial macrophages by
immune complexes, leading to decreased production
of proinflammatory cytokines.
Despite their similar effects on SF macrophages,
IL-13, unlike IL-4, did not induce the proliferation of SF
T cells. These data are consistent with previous studies
indicating that IL-13 does not induce proliferation of
normal human PB T cells or T cell clones (31,46).
However, these results are somewhat contradictory to
those of a recent study demonstrating that IL-4 and
TL-13 activate the same nuclear transcription factor in
highly purified human T cells (47). Thus, it remains
possible that T cells express functional IL-13 receptor
complex, but the IL-13-induced signaling pathway may
lack components that are required for the induction of T
cell proliferation. T cells specific for the as-yet-unknown
antigen appear to be important in the initiation and
maintenance of rheumatoid synovitis (14-16). Therefore, the stimulatory effects of IL-4 on the proliferation
of antigen-specific T cells may potentiate the inflammatory responses in the synovium. On the other hand, it has
been shown that IL-4 can induce the generation of Th2
cells during the initial antigen presentation to naive T
cells, whereas IL-13 is ineffective (54). Although the
roles of T h l and Th2 cytokines in systemic autoimmune
diseases, such as RA, are not clearly characterized,
Th2-type T cell response has been shown to attenuate
organ-specific autoimmune disorders in animal models
(20). Therefore, it remains to be shown whether the
lack of a T cell growth-promoting effect by IL-13 is
more important in attenuating the synovial inflammation than the ability of IL-4 to induce the differentiation
of Th2 cells.
Our findings demonstrate the presence of IL-13
in the joints of patients with RA. In addition, our results
indicate that IL-4 and IL-13 are not coexpressed in all
patients, suggesting separate roles for IL-4 and IL-13 in
controlling the immune responses in the synovium.
Moreover, the fact that IL-13 is unable to induce T cell
IL-13 IN RHEUMATOID ARTHRITIS
activation suggests a major difference in the effects of
IL-4 and IL-13 on synovial inflammation, confirmation
of which awaits the results of in vivo studies. Nevertheless, the demonstration that exogenous IL-13 inhibits the
production of proinflammatory cytokines by SFMC suggests that endogenous IL-13 is produced at concentrations that are not sufficient to optimally suppress
cytokine production in rheumatoid synovium, and
indicates that it may have therapeutic potential in the
treatment of patients with RA.
ACKNOWLEDGMENTS
We thank Satu Kling and Marju Niskala for technical
assistance. We are grateful to Dr. J a n E. d e Vries for valuable
discussions and t o Drs. Benjamin G. Cocks and Bruce Bennett
for their kind help. Dr. Riitta Saario is acknowledged for
providing patient samples.
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