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Interleukin-17 up-regulation of nitric oxide production in human osteoarthritis cartilage.

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ARTHRITIS & RHEUMATISM
Vol. 40, No. 6, June 1997, pp 1050-1053
0 1997, American College of Rheumatology
1050
INTERLEUKIN-17 UP-REGULATION OF NITRIC OXIDE PRODUCTION
IN HUMAN OSTEOARTHRITIS CARTILAGE
MUKUNDAN G. A n U R , RAJESH N. PATEL, STEVEN B. ABRAMSON, and ASHOK R. AMIN
Objective. To examine the effect of human
interleukin-17 (IL-17) on nitric oxide (NO) production
in human osteoarthritis (OA) cartilage under ex vivo
conditions.
Methods. OA cartilage from patients undergoing
knee replacement surgery was used in explant assays to
assess the effect of IL-17. NO production was measured
by estimating the stable NO metabolite, nitrite, in
conditioned medium.
Results. IL-17 augmented the spontaneous production of nitric oxide. This augmentation was sensitive
to cycloheximide and pyrrolidine dithiocarbamate, but
not to dexamethasone or soluble IL-1 receptor.
Conclusion. IL-17 augments nitric oxide production in OA cartilage via nuclear factor KB activation, but
independently of IL-lP signaling.
Interleukin-17 (IL-17, also designated CTLA-8)
produced by activated T cells shows -57% homology to
open reading frame 13 of a lymphotropic virus, Helpesvirus saimin' (HVS13) (1). IL-17 has been shown to
activate the transcription factor nuclear factor KB (NFKB), to induce expression of granulocyte colonystimulating factor (G-CSF), IL-6, IL-8, prostaglandin E,
(PGE,), and surface intercellular adhesion molecule 1in
fibroblasts, and to enhance the proliferation of T cells
induced by suboptimal costimulation with phytohemagglutinin (1). Overexpression of nitric oxide (NO) has
been implicated in various pathophysiologic conditions
Supported by Hospital for Joint Diseases institutional funds.
Mukundan G. Attur, PhD, Rajesh N. Patel, PhD: Hospital for
Joint Diseases, New York, New York; Steven B. Abramson, MD,
Ashok R. Amin, PhD: Hospital for Joint Diseases, and New York
University Medical Center, New York, New York.
Address reprint requests to Ashok R. Amin, PhD, Department of Rheumatology Research Laboratory, Room 1600, Hospital
for Joint Diseases, 301 East 17th Street, New York, NY 10003.
Submitted for publication October 21, 1996; accepted in
revised form January 24, 1997.
(2), including human rheumatoid arthritis (RA) and
osteoarthritis (OA) (3-5). We have recently reported
that human OA cartilage expresses a nitric oxide synthase (OA-NOS) that spontaneously releases NO under
ex vivo conditions (3,6). This spontaneous release can be
augmented by cytokines such as IL-lP and tumor necrosis factor a (TNFa). The spontaneous (or cytokineaugmented) release of NO and PGE, by human OA
cartilage is sensitive to the NF-KBinhibitor pyrrolidine
dithiocarbamate (PDTC) and to cycloheximide (3,6). In
the present study, we investigated the effect of IL-17 on
NO production by human OA cartilage.
PATIENTS AND METHODS
Reagents and cell lines. Protease inhibitors, dexamethasone, cycloheximide, and PDTC were obtained from Sigma
(St. Louis, MO), human IL-lP from Fisher Scientific (Pittsburgh, PA); and p-monomethyl-L-arginine monoacetate (LNMA) from Cyc10,pss BioChemical (Salt Lake City, UT).
Recombinant human IL-17 was purchased from R & D
Systems (Minneapolis, MN). Purified recombinant soluble
IL-1 receptor (rsIL-1R) (type I) expressed in CHO cells was
provided by Immunex (Seattle, WA). The specific activity of
the rsIL-1R was 2.0 X lo4 units/mg (7).
Procurement of human cartilage. Cartilage slices were
obtained from the knees of patients (age 50-70 years) with
advanced OA who were undergoing knee replacement surgery.
The OA patients had not taken steroids or nonsteroidal
antiinflammatory drugs for at least 2 weeks prior to surgery.
OA-explant assay. Knee articular cartilage from the
OA patients was cut into 3-mm discs, and 4-6 discs were
placed, in triplicate or quadruplicate, in a 24-well plate in 2 ml
F-12 medium in the presence or absence of various modulators, as previously described (3).
Determination of nitrite. NO production was measured by estimating the stable NO metabolite, nitrite, in
conditioned medium, by a modified Griess reaction (8). Values
are expressed as
nitrite released per 100 mg cartilage.
RESULTS
In view of the apparent central role of NF-KBin
both OA-NOS expression and IL-17 signaling, we tested
IL-17 AND NITRIC OXIDE PRODUCTION IN OA CARTILAGE
Table 1. Effect of interleukin-17 (IL-17) on nitric oxide production
in osteoarthritis (OA) cartilage*
____
Modulator
Nitrite (pM)
Control
L-NMA
IL-17 (30 ngiml)
IL-17 (60 ngirnl)
IL-17 (90 ngiml)
IL-1p (5 ngiml)
2.9 2 1.3t
0.2 2 0.2
4.7 i 0.6
4.5 i 1.5
4.2 2 0.5
8.4 i 0.9
* Affected knee articular cartilage from OA patients was cut into
3-mm disc; 4-6 discs were placed in organ culture (in triplicate) in 2 ml
F-12 medium in the presence or absence of various modulators.
Production of nitric oxide was assayed at 24 hours. Data are from 1 of
2 similar experiments, and are expressed as the mean 2 SD phi’ of
nitrite released per 100 ng cartilage (n = 3).
t P < 0.0083 versus IL-lptreated cartilage; P < 0.05 versus IL-17treated cartilage (30, 60, and 90 ngiml); P < 0.002 versus p monomethyl-L-arginine (L-NMA)-treated cartilage.
the effect of IL-17 on expression of OA-NOS. The
median effective dose (ED,,) of recombinant IL-17 used
in this study was 30 ng/ml, based on the ability of IL-17
at this dose to induce IL-6 production in BALB/c-3T3
cells. This was similar to previous findings in human
synovial fibroblasts, where the ED,, for IL-6 production
was 50 ngml (9). We therefore studied IL-17 at 30, 60,
and 90 ng/ml to examine its effect on the spontaneous
release of NO in OA cartilage under ex vivo conditions.
A concentration of 30 ng/ml of IL-17 was found to be
sufficient to significantly augment NO production in OA
cartilage (Table 1).
OA cartilage, as previously observed, released
NO spontaneously in the medium (24-72 hours) under
ex vivo conditions (Table 2). Addition of IL-1p or IL-17
significantly augmented the release of NO at 24-72
hours in 2 different experiments using cartilage from
separate patients. The addition of cycloheximide,
L-NMA (NOS inhibitor), or PDTC (NF-KBinhibitor) to
IL-17-containing organ cultures reduced the release of
NO to levels below those of control cultures, while
dexamethasone had no significant effect. We have recently observed that rsIL-lR, but not soluble TNFa
receptor, when added to OA organ cultures, can inhibit
the spontaneous release of NO by at least 50%, indicating that autocrine/endogenous IL-1p modulates the
production of NO (Attur MG et al: unpublished data).
Addition of rsIL-1R to cultures treated with IL-17 did
not have any significant effect on the NO release, but, as
expected, did attenuate the IL-1P-induced (and partially, but significantly, the IL-1p + lipopolysaccharide
+ TNFa-induced) NO production in the same experiments (Table 3). These observations indicate that the
1051
effects of IL-17 on N O production in OA cartilage may
be independent of IL-1p signaling.
DISCUSSION
Our studies indicate that IL-17, like IL-lp, is
responsible for exacerbating NO production in OA
Table 2. Effect of IL-17 on nitric oxide production by human OA
cartilage under ex vivo conditions*
Nitrite (pM) in medium
Modulator
24 hours
Experiment 1
Control
0.3 2 0.3t
IL-lp (5 ngiml)
3.7 t 1.4
IL-17 (30 ngiml)
1.8 i 0.3$
<o. 1
IL-17 + cycloheximide
(1 PdmU
IL-17 + dexamethasone 1.8 t 0.9
(1 pM)
<0.1
IL-17 + PDTC (30 /JAW)
1.3 2 0.8
IL-17 + rsIL-1R
(10 Pgiml)
Experiment 2
Control
0.8 t 0.511
8.5 i 2.4
IL-10 (5 ngiml)
4.1 t 1.0#
IL-17 (30 ng/ml)
IL-17 + cycloheximide
0.0 t 0.0
(1
IL-17 + dexamethasone 2.6 2 0.5
(1 /JAW)
IL-17 + PDTC (30 /hf)0.4 t 0.3
IL-17 + L-NMA
1.0 t 1.0
(500 P W
48 hours
72 hours
0.7 t 0.5t
11.0 +- 3.9
6.3 i 1.4$
0.2 t 0.1
1.2 t 0.5t
14.6 i 4.5
7.0 i 2.24
0.5 t 0.1
4.6 t 2.2
7.4 2 2.3
0.4 i 0.5
5.1 2 2.4
0.7 i 0.6
7.2 i 2.9
3.1 t 0.611
8.6 t 1.97
21.4 i 4.3
26.9 i 3.5
8.9 i 2.3** 14.4 + 3.7tt
0.2 t 0.0
0.7 t 0.2
7.1 2 0.1
11.3 2 2.3
0.5 t 0.5
1.3 2 1.1
1.0 i 0.5
2.0 t 0.9
*Affected knee articular cartilage from OA patients was cut into
3-mm discs; 4-6 discs were placed in organ culture (in triplicate) in 2
ml F-12 medium in the presence or absence of various modulators.
Cartilage from 2 separate patients was used in experiments 1 and 2.
Data are expressed as the mean i SD pA4 of nitrite released per 100
ng cartilage (n = 3). PDTC = pyrrolidine dithiocarbamate; rsIL-1R =
recombinant soluble IL-1 receptor. See Table 1 for other definitions.
t P < 0.001 versus IL-lptreated cartilage.
$ P < 0.0001 versus control, versus IL-17 + cycloheximide-treated
cartilage, and versus IL-17 + PDCT-treated cartilage.
9 P < 0.0012 versus control; P < 0.0001 versus IL-17 + cycloheximidetreated cartilage and versus IL-17 + PDTC-treated cartilage.
7 P < 0.001 versus IL-lp-treated cartilage.
# P < 0,0001 versus control, versus IL-17 + cycloheximide-treated
cartilage, and versus IL-17 + PDTC-treated cartilage; P < 0.001
versus IL-17 + L-NMA-treated cartilage; P < 0.02 versus IL-17 +
dexamethasone-treated cartilage.
* * P < 0,0001 versus control, versus IL-17 + cycloheximide-treated
cartilage, and versus IL-17 + PDTC-treated cartilage; P < 0.001
versus IL-17 + L-NMA-treated cartilage.
tt P < 0.03 versus control; P < 0.0001 versus IL-17 + cycloheximidetreated cartilage and versus IL-17 + PDTC-treated cartilage; P <
0.001 versus IL-17 + L-NMA-treated cartilage.
ATTUR ET AL
1052
Table 3. Effect of recombinant soluble IL-1 receptor (rsIL-1R) on nitric oxide production induced by
IL-17 in human OA cartilage under ex vivo conditions*
Nitrite
Modulators
Experiment 1
Control
IL-1p
IL-lp + rsIL-1R
IL- 17
IL-17 + rsIL-1R
Experiment 2
Control
IL-lp
IL-1p + rsIL-1R
IL- 17
IL-17 + rsIL-1R
LPS + IL-lp + TNFa
LPS + IL-1p + TNFa
+ rsIL-1R
( N )in medium
24 hours
48 hours
72 hours
1.1 t 0.4
4.3 t 1.27
1.7 t 0.9
2.2 t 0.67
2.0 t 1.7
5.2 t- 1.7
20.2 t- 5.8$
7.4 t 2.8
11.0 2 3.0#
11.8 t 6.8
6.6 t 2.2
31.0 ? 8.35
9.2 t 2.5
13.3 t 4.6**
10.9 i- 4.6
0.9 t 0.677
9.3 t- 1.5$$
4.6 2 1.0
8.4 t 2.5
6.7 t 1.3
10.1 t 2.7
7.3 t 1.7
1.2 t 0.477
27.4 2 2.959
10.6 t 2.5
21.9 t- 5.5
22.4 t 3.4
26.7 t 3.9
18.9 2 3.877
5.4 t 1.777
35.3 t 4.055
11.6 t 2.5
27.0 t 7.8
29.2 t 6.8
37.8 t 5.6
24.4 t 4.2##
* Affected knee articular cartilage from OA patients was cut into 3-mm discs; 4-6 discs were placed in
organ culture (in triplicate) in 2 ml F-12 medium in the presence or absence of various modulators.
Cartilage from 2 separate patients was used in experiments 1 and 2. Data are expressed as the mean t SD
f l of nitrite released per 100 ng cartilage (n = 3). LPS = lipopolysaccharide; TNFa = tumor necrosis
factor a. See Table 1 for other definitions.
7 P < 0.001 versus control; P < 0.007 versus rsIL-1 R-treated cartilage.
$ P < 0.001 versus control; P < 0.004 versus rsIL-1R-treated cartilage.
5 P < 0.001 versus control and versus rsIL-lR-treated cartilage.
7 P < 0.01 versus control.
# P < 0.008 versus control.
* * P < 0.02 versus control.
$7 P < 0.0001 versus IL-lp-treated cartilage and versus LPS + IL-lp + TNFa-treated cartilage; P <
0.001 versus IL-17-treated cartilage.
$$ P < 0.001 versus rsIL-1R-treated cartilage.
$0 P < 0.0001 versus rsll-1R-treated cartilage.
77 P < 0.01 versus LPS + IL-1p + TNFcu-treated cartilage.
## P < 0.004 versus LPS + IL-1B + TNFa-treated cartilage.
cartilage. The levels of nitrite accumulated by IL-17stimulated cartilage were generally lower than those
observed with IL-lp, even in experiments in which
higher concentrations of IL-17 were used, thus showing
a marginal difference in the sensitivity of OA-NOS to
these 2 mediators of inflammation. These experiments
also suggest that IL-17-dependent augmentation of NO
production requires de novo protein synthesis and
NF-KB activation in human OA cartilage. Furthermore,
the IL-17-induced NO production in these explants is
not sensitive to the addition of dexamethasone, whereas
we have recently observed >90% inhibition of spontaneously released PGE, production in dexamethasonetreated cartilage (6).
IL-17 up-regulates cytokines such as IL-6, GCSF, and IL-8 in synovial cells (9). These cytokines have
been detected in OA and RA synovial fluids and are
reported to be released also by chondrocytes (10-12). It
is tempting to speculate that the role of IL-17 in
inflammatory arthropathies such as RA may be more
pronounced because the infiltrated T cells in the joints
releasing IL-17 may not only up-regulate other cytokines
in the joints, but may also directly up-regulate NO
production in the cartilage. These initial studies of IL-17
constitute the proverbial “tip of the iceberg,” since IL-17
is assumed to regulate other cytokines. The fact that
IL-17 regulates other pleiotropic secondary signaling
messenger molecules, such as NO (2) independent of
cytokines such as IL-lp, expands its array of functions and
pleiotropism, especially in chondrocytes, where overexpression of NO has been implicated in chondrocyte dysfunction (5). Analysis of the effects of blocking IL-17
activity, for example by use of the recently described
rsIL-17 receptor (13), will be of particular interest.
IL-17 AND NITRIC OXIDE PRODUCTION IN OA CARTILAGE
ACKNOWLEDGMENTS
We would like t o thank Immunex Corporation for
providing the slL-1 R for this study. We a r e grateful to Drs. G.
Thakker and I. Patel for their contributions during the course
of the studv and for their constructive criticism.
REFERENCES
1. Broxmeyer HE: Is interleukin 17, an inducible cytokine that
2.
3.
4.
5.
6.
stimulates production of other cytokines, merely a redundant
player in a sea of other biomolecules? J Exp Med 183:2411-2415,
1996
Schmidt HHHW, Walter U: Nitric oxide at work. Cell 78:919-925,
1994
Amin AR, DiCesare P, Vyas P, Attur M, Tzeng E, Billiar TR,
Stuchin SA, Abramson SB: The expression and regulation of nitric
oxide synthase in human osteoarthritis-affected chondrocytes:
evidence for upregulated neuronal nitric oxide synthase. J Exp
Med 182:2091-2102, 1995
Sakurai H, Kohsaka H, Liu MF. Higashiyama H, Hirata Y, Kanno
K, Saito I, Miyasaka N: Nitric oxide production and inducible
nitric oxide synthase expression in inflammatory arthritides. J Clin
Invest 96:2357-2363, 1995
Clancy RM, Amin AR, Abramson SB: The role of nitric oxide in
inflammation. Submitted for publication
Amin AR, Attur M, Patel RN, Thakker GD, Marshall PJ, Rediske
J, Stuchin SA, Patel IR, Abramson SB: Superinduction of
cyclooxygenase-2in human osteoarthritis-affected cartilage: influence of nitric oxide. J Clin Invest (in press)
1053
7. Dower SK, Wignall JM, Schooley K, McMahan CJ, Jackson JL,
Prickett KS, Lupton S, Cosman D, Sims JE: Retention of ligand
binding activity by the extracellular domain of the IL-I receptor.
J lmmunol 142:4314-4320, 1989
8. Gilliam MB, Sherman MP, Griscavage JM, Ignarro U: A spectrophotometric assay for nitrate using NADPH oxidation bv Asoergilius nitrate reduciase. Anal Biochim 212:359-365, 199;
9. Fossiez F, Djossou 0, Chomarat P, Flores-Romo L, Ait-Yahia S,
Maat C, Pin JJ, Garrone P, Garcia E, Saeland S, Blanchard D,
Gaillard C, Das Mahapatra B, Rouvier E, Golstein P, Banchereau
J, Lebecque S: T cell interleukin-17 induces stromal cells to
produce proinflammatory and hematopoietic cytokines. J Exp
Med 183:2593-2603, 1996
10. Schlaak JF, Pfers I, zum Buschenfelde KHM, Marker HE: Different cytokine profiles in the synovial fluid of patients with osteoarthritis, rheumatoid arthritis and seronegative spondyloarthropathies. Clin Exp Rheumatol 14:155-162, 1996
11. Recklies AD, Golds EE: Induction of synthesis and release of
interleukin-8 from human articular chondrocytes and cartilage
explants. Arthritis Rheum 35:1510-1519, 1992
12. Venn G , Nietfeld JJ, Duits AJ, Brennan FM, Arner E, Covington
M, Billingham MEJ, Hardingham TE: Elevated synovial fluid
levels of interleukin-6 and tumor necrosis factor associated with
early experimental canine osteoarthritis. Arthritis Rheum 36319826, 1993
13. Yao Z, Fanslow WC, Seldin MF, Rousseau AM, Painter SL,
Comeau MR, Cohen JI, Spriggs MK: Herpesvirus saimiri encodes
a new cytokine, IL-17, which binds to a novel cytokine receptor.
Immunity 3311-821, 1995
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