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Mediation of the proinflammatory cytokine response in rheumatoid arthritis and spondylarthritis by interactions between fibroblast-like synoviocytes and natural killer cells.

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ARTHRITIS & RHEUMATISM
Vol. 58, No. 3, March 2008, pp 707–717
DOI 10.1002/art.23264
© 2008, American College of Rheumatology
Mediation of the Proinflammatory Cytokine Response in
Rheumatoid Arthritis and Spondylarthritis by Interactions
Between Fibroblast-like Synoviocytes and Natural Killer Cells
Antoni Chan,1 Andrew Filer,2 Greg Parsonage,2 Simon Kollnberger,1
Roger Gundle,3 Christopher D. Buckley,2 and Paul Bowness4
IL-1␤, and IL-15, was increased in cocultures of NK
cells and FLS, particularly in those from RA and SpA
patients. Production of interferon-␥, RANTES, and
matrix metalloproteinase 3 (MMP-3) by NK cell and
FLS coculture was greatest in SpA patients. Surface
expression of IL-15 on FLS was significantly increased
in SpA and RA patients, but not OA patients. Blockade
with an IL-15 monoclonal antibody resulted in increased apoptosis of NK cells.
Conclusion. FLS promote the migration, activation, and survival of NK cells. The interaction of NK
cells with FLS results in increased IL-15 expression by
FLS and the production of proinflammatory chemokines, cytokines, and MMPs, which may contribute to
joint inflammation. This response was much more
marked in SpA and RA patients as compared with OA
patients.
Objective. Fibroblast-like synoviocytes (FLS) are
potentially directly involved in the propagation of inflammation. We have previously shown evidence of an
expanded activated population of natural killer (NK)
cells in spondylarthritis (SpA) patients. In the present
study, we sought to determine whether the interaction
between NK cells and FLS from SpA patients results in
a proinflammatory response.
Methods. Autologous NK cells and FLS were
obtained from 6 patients with SpA, 4 patients with
rheumatoid arthritis (RA), and 8 patients with osteoarthritis (OA). Physical interactions between NK cells and
FLS were studied by time-lapse phase-contrast microscopy. Fluorescence-activated cell sorting was used to
study the activation, proliferation, and survival of NK
cells in contact with FLS. Cytokine and stromal factor
production were measured by a multiple cytokine bead
assay.
Results. NK cells both adhered to and migrated
beneath the FLS monolayer (pseudoemperipolesis).
FLS from SpA and RA patients supported increased
pseudoemperipolesis, activation, cytokine production,
and survival of NK cells. The production of proinflammatory cytokines, including interleukin-6 (IL-6), IL-8,
The synovial membrane lining comprises 2 dominant cell populations. These consist of macrophage-like
synoviocytes (type A synoviocytes) and fibroblast-like
synoviocytes (FLS; type B synoviocytes), the latter distinguished by their surface marker expression profile (1).
Two-thirds of native synoviocytes are FLS, which function to provide structural integrity and to maintain the
articular surface in health (2). Recent evidence shows
that FLS are not passive players in the immune response
(3) and may regulate the switch from acute resolving
inflammation to chronic persistent inflammation (4).
Little is known of the role of FLS in spondylarthritis (SpA). In rheumatoid arthritis (RA), FLS are
involved in the recruitment, activation, and survival of T
cells in the joint (5). FLS participate actively in cell
recruitment by producing chemokines, including monocyte chemotactic protein (MCP), interleukin-8 (IL-8),
RANTES, and IL-16 (6,7). Retention of T cells within
the joint is aided by the production of the chemokine
Supported by the Arthritis Research Campaign and the
Medical Research Council, UK.
1
Antoni Chan, PhD, MRCP, Simon Kollnberger, PhD: John
Radcliffe Hospital, Oxford, UK; 2Andrew Filer, MBChB, PhD, Greg
Parsonage, PhD, Christopher D. Buckley, MBBS, FRCP: University of
Birmingham, Birmingham, UK; 3Roger Gundle, DPhil: Nuffield Orthopaedic Centre, Oxford, UK; 4Paul Bowness, MB BChir, DPhil:
John Radcliffe Hospital, Oxford, and Nuffield Orthopaedic Centre,
Oxford, UK.
Address correspondence and reprint requests to Antoni
Chan, PhD, MRCP, Medical Research Council Human Immunology
Unit, Weatherall Institute of Molecular Medicine, John Radcliffe
Hospital, Headley Way, Headington, Oxford OX3 9DS, UK. E-mail:
achan@hammer.imm.ox.ac.uk or antoni.chan@royalberkshire.nhs.uk.
Submitted for publication March 27, 2007; accepted in revised
form November 26, 2007.
707
708
CHAN ET AL
stromal cell–derived factor 1 (CXCL12) by FLS and by
the expression of its receptor CXCR4 on infiltrating T
cells (8,9). The prolonged survival of T cells in the
rheumatoid joint is supported principally by type I
interferons (IFNs) produced by FLS (10). FLS also
produce IL-6, which may regulate the switch from innate
to acquired immunity through differential control of
leukocyte recruitment, activation, and apoptosis (11,12).
Natural killer (NK) cells are effector cells of the
innate immune system; they comprise 10–15% of peripheral blood lymphocytes (13). They express CD56, but
not B cell or T cell markers such as CD3 (14). NK cells
play an important role in the defense against virusinfected and tumor cells (15). NK cells bear a number of
cytokine receptors, including those for IL-2, IL-12, IL15, IL-21, and IFN␣/␤. These cytokines can promote NK
proliferation, cytotoxicity, and production of cytokines
such as IFN␥, tumor necrosis factor ␣ (TNF␣),
granulocyte–macrophage colony-stimulating factor
(GM-CSF), IL-5, IL-13, macrophage inflammatory protein 1␣/␤ (MIP-1␣/␤), and RANTES (16–18). We have
recently shown an expansion of NK cells bearing the
killer cell immunoglobulin-like receptor 3DL2 (KIR3DL2) in the peripheral blood and synovial fluid of SpA
patients (19). Ex vivo, KIR-3DL2⫹ NK cells from SpA
patients had an activated phenotype and were protected
from activation-induced cell death. We hypothesized
that NK cells may interact with resident FLS in the joint
through cell-to-cell contact as well as with cytokines and
that this could result in a proinflammatory response.
In this study, we found that ex vivo, isolated NK
cells physically interact with FLS. This interaction results
in mutual activation and production of proinflammatory
cytokines and other factors that may contribute to joint
damage, including IFN␥, IL-6, IL-8, GM-CSF, MCP-1,
and matrix metalloproteinase 3 (MMP-3). Furthermore,
we found evidence of a significant role of IL-15 in this
interaction.
PATIENTS AND METHODS
Patients and samples. Synovial tissue samples were
collected from the knee joints of 6 SpA, 4 RA, and 8
osteoarthritis (OA) patients who were undergoing arthroplasty
or synovectomy. SpA was defined according to the European
Spondylarthropathy Study Group criteria (20). Four SpA
patients fulfilled the modified New York criteria for ankylosing
spondylitis (AS) (21). Of the 4 AS patients, 3 were HLA–B27
positive (HLA–B*2702/2705) by DNA typing. The remaining 2
SpA patients, both of whom were HLA–B27 positive, had
psoriatic arthritis. RA patients fulfilled the American College
of Rheumatology (ACR; formerly, the American Rheumatism
Association) revised criteria (22). OA of the knee was defined
according to the ACR criteria (23). The main demographic
and clinical data on the study patients are shown in Table 1.
This study had ethical approval from the Oxford
Radcliffe Trust local (COREC C00.114) and the South Birmingham Local Ethics Committee (LREC 5735).
Isolation of peripheral blood mononuclear cells
(PBMCs). Peripheral blood was collected into 50-ml polypropylene tubes (BD Falcon, Bedford, MA) prepared with 100 ␮l
(5,000 units) of sodium heparin. PBMCs were isolated by
centrifugation at 2,000 revolutions per minute for 20 minutes
using a Ficoll-Hypaque gradient (Lymphoprep; Nycomed
Pharma, Oslo, Norway). Cells at the interface of the 2 phases
were collected and washed twice with RPMI 1640 medium
(Gibco BRL, Grand Island, NY), and cells were resuspended
in R10 (RPMI 1640 medium supplemented with 2 mM
L-glutamine, 100 IU of penicillin/streptomycin, and 10% fetal
calf serum [FCS]). Both fresh and cryopreserved cells were
compared by repeated stainings, and consistent results were
obtained, with a ⬍5% difference (19).
FLS and NK cell culture. Fine strips of synovial tissue
⬍1 mm3 in volume were suspended in 10 ml of wash buffer
(RPMI 1640 and 20 mM HEPES). After centrifugation at 200g
for 10 minutes, the supernatant was discarded and the pellet
resuspended in wash buffer. The wash step was repeated 2
more times. The pellet was resuspended in 10 ml of digestion
buffer (RPMI 1640, 20 mM HEPES, and 0.2% collagenase
type 1A) and incubated for 4–5 hours at 37°C, with vigorous
shaking. The digestion mixture was then centrifuged at 200g
for 10 minutes, the supernatant was discarded, and the pellet
was resuspended in 10 ml of fresh medium (RPMI 1640, 20%
FCS, 1% glutamine, 1% penicillin/streptomycin, 1% nonessential amino acids, and 1% sodium pyruvate; Sigma-Aldrich, St.
Louis, MO) and cultured to confluence as previously described
(24,25). FLS had characteristic morphology and expressed
fibronectin and prolyl-4-hydroxylase, but not CD1, CD3,
CD19, CD31, CD68, CD80, CD86, von Willebrand factor, or
cytokeratin (26). FLS were used between passages 3 and 5.
NK cells were enriched from PBMCs using a magneticactivated cell sorting NK-negative isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity of the cells was
determined before they were used, and ⬎99% were found to
be CD3–,CD56⫹ by flow cytometry.
NK cells and FLS coculture conditions. FLS were
passaged and added to 96-well flat-bottomed plates (BD
Falcon) at a density of 3,000 cells/well. Experiments were
performed with confluent FLS prepared 24 hours before
contact. Once confluence was confirmed by microscopy, the
fibroblasts were washed twice in phosphate buffered saline
(PBS) and cocultured for 7 days with 1 ⫻ 105 NK cells in R10
(RPMI 1640 medium supplemented with 2 mM L-glutamine,
100 IU of penicillin/streptomycin, and 10% FCS). We have
previously shown that NK cell differentiation and proliferation
occur after 5 days of NK cell/FLS coculture, with many NK
cells undergoing apoptosis after 10 days of coculture without
the addition of cytokines (27). Therefore, analyses were performed on day 7. The control monolayers of FLS remained
confluent at 7 days in the absence of NK cells. All experiments
were performed in duplicate.
A 24-well Transwell system (Corning Costar, Cambridge, MA) was used in some coculture experiments. This
system consists of 2 compartments separated by a porous
MUTUAL STIMULATION OF FLS AND NK CELLS IN INFLAMMATORY ARTHRITIS
Table 1.
709
Demographic, laboratory, and clinical features of the study patients, by diagnostic group*
Age, mean (range) years
Sex, no. male/female
No. (%) HLA–B27⫹
No. of HLA–B27⫹ SpA patients
AS
PsA
BASDAI score, mean ⫾ SD
No. (%) RF⫹
DAS28 score, mean ⫾ SD
No. (%) taking DMARDs
No. taking MTX
No. taking SSZ
No. (%) taking corticosteroids
No. taking 5 mg/day of prednisolone
Previous anti-TNF␣ therapy†
No. who took infliximab
No. who took etanercept
No. who took adalimumab
SpA patients
(n ⫽ 6)
RA patients
(n ⫽ 4)
OA patients
(n ⫽ 8)
39.7 (28–53)
4/2
5 (83)
40.8 (28–59)
1/3
0 (0)
46.7 (31–77)
4/4
0 (0)
3
2
3.9 ⫾ 1.0
0 (0)
–
2 (33)
1
1
0 (0)
0
0 (0)
0
0
0
–
–
–
3 (75)
4.1 ⫾ 1.1
4 (100)
4
2
1 (25)
1
1 (25)
1
0
0
–
–
–
0 (0)
–
0 (0)
0
0
0 (0)
0
0 (0)
0
0
0
* SpA ⫽ spondylarthritis; RA ⫽ rheumatoid arthritis; OA ⫽ osteoarthritis; AS ⫽ ankylosing spondylitis;
PsA ⫽ psoriatic arthritis; BASDAI ⫽ Bath Ankylosing Spondylitis Disease Activity Index; RF ⫽
rheumatoid factor; DAS28 ⫽ Disease Activity Score in 28 joints; DMARDs ⫽ disease-modifying
antirheumatic drugs; MTX ⫽ methotrexate; SSZ ⫽ sulfasalazine.
† None of the patients were receiving an anti–tumor necrosis factor ␣ (anti-TNF␣) agent at the time of
the study.
matrix (0.4 ␮m), which allows the exchange of soluble factors
while preventing direct contact. FLS were grown to confluence
in the bottom well, and NK cells were either added to the same
well (allowing contact) or to the top well (avoiding contact).
Anti-human IL-6 monoclonal antibody (mAb) and
anti-human IL-15 mAb (both from R&D Systems, Abingdon,
UK) blocking antibodies and isotype control antibody (BD
PharMingen, San Jose, CA) were added at a concentration of
10 ␮g/ml to the FLS for 30 minutes at 4°C. Neutralizing
polyclonal goat IgG anti–IL-15R␣ antibody (R&D Systems) or
control goat IgG was used at 1 ␮g/ml and was incubated with
NK cells for 30 minutes at 4°C. NK cells were subsequently
added to the FLS.
Antibodies, fluorescence-activated cell sorter (FACS)
analysis, and immunostaining. NK cells were washed in PBS
in the presence of 1% heat-inactivated FCS and 0.1% sodium
azide (weight/volume) and incubated for 30 minutes on ice
with saturating amounts of the following directly conjugated
anti-human mAb: CD3, CD16, CD94, and CD56 (Serotec,
Oxford, UK). The following directly conjugated anti-human
mAb were also used in this study: CD68, CD69, CD80, CD86,
CD90, HLA–DR, vascular cell adhesion molecule 1, NKp46,
NKp30, and IFN␥ (BD Biosciences, San Jose, CA) and IL-15
(R&D Systems). Cells were washed twice after incubation with
mAb. Cells were fixed in 1% paraformaldehyde and analyzed
with a FACSCalibur cell sorter using CellQuest software
(Becton Dickinson, Mountain View, CA). Intracellular cytokine staining was used to detect IFN␥ release using a Cytofix/
Cytoperm kit (BD Biosciences). As positive controls for intracellular cytokine staining, phorbol myristate acetate (10 ng/ml)
and ionomycin (1 ng/ml) were used (Sigma-Aldrich, Dorset,
UK).
Pseudoemperipolesis assays. Fibroblasts were seeded
onto glass chamber slides at a density of 1 ⫻ 105/well and
cultured for 3 days. Prior to coculture, the fibroblast layer was
washed, and 400 ␮l of R10 medium was added. A 100-␮l
volume of autologous NK cells (2.5 ⫻ 105) was added to the
well, and the coculture was gently mixed, then incubated under
stasis for a period of 2 hours. Pseudoemperipolesis was assessed by counting phase-dark cells in 3 independent fields and
was expressed as a percentage of the total input NK cells.
Phase-dark cells were cells that had migrated into and under
the fibroblast layer; phase-light cells were cells that either
remained nonadherent or adhered to the surface of the
fibroblast layer. A Zeiss Axiovert 200 microscope system with
settings for phase contrast was used (Carl Zeiss Instruments,
Welwyn Garden City, UK). Images were captured and combined into time-lapse movies using a Hamamatsu C4742-95
camera and Simple PCI software (Hamamatsu Photonics,
Welwyn Garden City, UK).
Survival assays. NK cells (1 ⫻ 105 cells) and FLS
(3,000 cells) were cultured for 7 days in 200 ␮l of R10 in
flat-bottomed 96-well plates. Cells were harvested and stained
for annexin V and 7-aminoactinomycin D (7-AAD) according
to the manufacturer’s instructions (BD Biosciences) and then
analyzed by FACS. Annexin V–negative, 7-AAD–negative
cells were designated fully viable, annexin V–positive, 7-AAD–
negative cells were designated as undergoing early apoptosis
with membrane integrity present, and annexin V–positive,
7-AAD–positive cells were designated as end-stage apoptotic
cells committed to death.
Labeling and analysis with 5,6-carboxyfluorescein diacetate N-succinimidyl ester (CFSE-DA). NK cells were labeled with 5 ␮M CFSE-DA (Molecular Probes, Eugene, OR)
710
CHAN ET AL
RESULTS
High levels of NK cell pseudoemperipolesis in
RA and SpA FLS. We have previously shown that
fibroblasts taken from different sites interact with T cells
in a different manner, such that synovial fibroblasts, but
not skin fibroblasts, can support T cell pseudoemperipolesis (9). However, no comparison has yet been made
between fibroblasts derived from the same anatomic
sites but representing different diseases. Time-lapse
phase-contrast microscopy showed that NK cells exhibited pseudoemperipolesis when in contact with autologous FLS from individuals with OA, SpA, and RA
(Figure 1). NK cells from SpA and RA patients exhib-
Figure 1. High levels of natural killer (NK) cell pseudoemperipolesis
in fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA)
and spondylarthritis (SpA) patients, but not osteoarthritis (OA) patients, as determined by time-lapse phase-contrast microscopy.
Pseudoemperipolesis was assessed by counting phase-dark cells (those
that migrated into and under the fibroblast layer) in 3 independent
fields. Results were expressed as a percentage of the total input NK
cells cocultured with autologous FLS. Values are the mean and SD of
triplicate determinations of samples from 6 SpA, 4 RA, and 4 OA
patients. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. ns ⫽ not significant.
at 37°C as described previously (28). After culture, the
CFSE-DA content and surface phenotype were simultaneously
analyzed by FACS. NK cells were stimulated with 100 IU/ml of
IL-2 (PeproTech, London, UK) as a positive control.
CFSE-DA was used to determine the number of divisions that
NK cells had undergone during culture. Cell division in
CFSE-DA–labeled NK cells was calculated based on the
sequential halving of fluorescence intensity in daughter cells;
thus, the cell numbers in each peak were divided by the
expected progeny for those divisions (divided by 2 for 1
division, by 4 for 2 divisions, by 8 for 3 divisions, etc.) as
described elsewhere (29).
Multiple cytokine bead assay. NK cells were cocultured with FLS, and supernatants were harvested on day 7 and
stored at –80°C before being assayed. Cytokine assays were
performed using the Bio-Plex (multiplex) cytokine assay according to the manufacturer’s instructions (Bio-Rad, Hercules,
CA) and as previously described (30). Cytokines in the supernatants were measured with the Bio-Plex suspension array
system instrument and were analyzed using Bio-Plex Manager
software (both from Bio-Rad).
Statistical analysis. Data were analyzed using Student’s 2-tailed t-test. When comparing more than 2 groups,
one-way analysis of variance was performed. The level of
significance was set at P ⬍ 0.05.
Figure 2. Coculture of natural killer (NK) cells and fibroblast-like
synoviocytes (FLS) from patients with spondylarthritis (SpA), rheumatoid arthritis (RA), and osteoarthritis (OA) and NK cell activation,
as determined by fluorescence-activated cell sorter analysis. A, Expression of CD69 on NK cells cultured in media only, with FLS, or with
FLS in the presence of interleukin-6 (IL-6)–blocking monoclonal
antibody (mAb). Results are from a representative patient of 6 SpA, 4
RA, and 8 OA patients evaluated. Shaded histograms show the
percentages of CD69⫹ NK cells; open histograms show NK cells
stained with isotype control antibody. B, Expression of HLA–DR on
NK cells cultured in media only, with FLS, or with FLS in the presence
of IL-6–blocking mAb. In all cases, autologous NK cells and FLS were
cocultured. Values are the mean and SD of cells from 6 SpA, 4 RA,
and 8 OA patients. ⴱ ⫽ P ⬍ 0.05.
MUTUAL STIMULATION OF FLS AND NK CELLS IN INFLAMMATORY ARTHRITIS
711
Table 2. Cytokine and stromal factor levels in cell culture supernatants, by diagnostic group*
Analyte concentration
NK cell ⫹ media
NK cell ⫹ FLS
FLS ⫹ media
Analyte†
SpA
RA
OA
SpA
RA
OA
SpA
RA
OA
IL-1␤
IL-2
IL-4
IL-5
IL-6
IL-7
IL-8
IL-10
IL-12
IL-13
IL-17
IL-15
TNF␣
IFN␥
GM-CSF
MIP-1␤
MCP-1
G-CSF
VEGF
RANTES
IP-10
bFGF
PDGF
EGF
MMP-3
MMP-9
MMP-13
1⫹
–
–
–
–
–
–
–
–
–
–
1⫹
–
1⫹
–
1⫹
–
–
1⫹
2⫹
–
–
–
–
–
1⫹
–
1⫹
–
–
–
–
–
–
–
–
–
–
–
–
1⫹
–
1⫹
–
–
1⫹
2⫹
–
–
–
–
–
1⫹
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1⫹
1⫹
–
–
–
–
–
1⫹
–
1⫹
–
–
1⫹
5⫹
–
6⫹
–
–
3⫹
–
3⫹
1⫹
3⫹
5⫹
2⫹
5⫹
2⫹
3⫹
4⫹
1⫹
–
–
1⫹
5⫹
2⫹
1⫹
1⫹
–
–
1⫹
5⫹
–
6⫹
–
–
2⫹
–
3⫹
1⫹
2⫹
5⫹
2⫹
5⫹
1⫹
3⫹
3⫹
1⫹
–
–
1⫹
4⫹
2⫹
1⫹
1⫹
–
–
1⫹
5⫹
–
5⫹
–
–
1⫹
–
1⫹
1⫹
1⫹
4⫹
1⫹
4⫹
1⫹
2⫹
1⫹
2⫹
–
–
1⫹
2⫹
2⫹
1⫹
1⫹
–
–
–
5⫹
–
1⫹
–
–
–
–
–
–
–
3⫹
–
2⫹
–
1⫹
–
–
–
–
1⫹
1⫹
–
–
1⫹
–
–
–
4⫹
–
1⫹
–
–
–
–
–
–
–
3⫹
–
2⫹
–
1⫹
–
–
–
–
1⫹
1⫹
–
–
1⫹
–
–
–
4⫹
–
1⫹
–
–
–
–
–
–
–
3⫹
–
2⫹
–
1⫹
–
–
–
–
1⫹
1⫹
–
–
* Supernatants obtained on day 7 from natural killer (NK) cells cultured in R10, NK cells cocultured with fibroblast-like
synoviocytes (FLS) in R10, and FLS cultured in R10 were analyzed for cytokine and stromal factors by multiple cytokine bead
assay. Duplicate wells were analyzed for each sample, and background levels in wells containing only R10 were subtracted.
Values are the mean of 3 independent experiments (each performed in duplicate) in 6 spondylarthritis (SpA), 4 rheumatoid
arthritis (RA), and 8 osteoarthritis (OA) patients. Results are defined as follows: – ⫽ undetectable or ⬍1 pg/ml, 1⫹ ⫽ 1–50
pg/ml, 2⫹ ⫽ 50.1–100 pg/ml, 3⫹ ⫽ 100.1–500 pg/ml, 4⫹ ⫽ 500.1–1,000 pg/ml, 5⫹ ⫽ 1,000.1–5,000 pg/ml, and 6⫹ ⫽
5,000.1–10,000 pg/ml.
† IL-1␤ ⫽ interleukin-1␤; TNF␣ ⫽ tumor necrosis factor ␣; IFN␥ ⫽ interferon-␥; GM-CSF ⫽ granulocyte–macrophage
colony-stimulating factor; MIP-1␤ ⫽ macrophage inflammatory protein 1␤; MCP-1 ⫽ monocyte chemotactic protein 1;
G-CSF ⫽ granulocyte colony-stimulating factor; VEGF ⫽ vascular endothelial growth factor; IP-10 ⫽ IFN␥-inducible 10-kd
protein; bFGF ⫽ basic fibroblast growth factor; PDGF ⫽ platelet-derived growth factor; EGF ⫽ epidermal growth factor;
MMP-3 ⫽ matrix metalloproteinase 3.
ited significantly increased levels of pseudoemperipolesis compared with those from OA patients (P ⬍ 0.05).
NK cell activation and interactions between NK
cells and RA or SpA FLS. To determine whether the NK
cell/FLS interaction could lead to activation of NK cells,
highly purified NK cells (⬎99% CD3–,CD56⫹) were
cocultured with autologous FLS for 24 hours. CD69 and
HLA–DR were used as surrogate markers of NK cell
activation (31). As shown in Figure 2, the expression of
CD69 and HLA–DR was increased on NK cells after
coculture with FLS. This was significantly higher in the
SpA and RA groups as compared with the OA group.
Thus, a mean ⫾ SD of 69.4 ⫾ 4.8% (n ⫽ 6) and 64.5 ⫾
6.9% (n ⫽ 4) of NK cells from the SpA and RA groups,
respectively, expressed CD69 when cocultured with FLS,
as compared with 51.7 ⫾ 4.8% (n ⫽ 8) of NK cells from
the OA group (P ⬍ 0.05 for both comparisons) (Figure
2A).
Significant increases in NK cell HLA–DR expression were also observed upon coculture with FLS, and
these were also more marked in cells from the SpA and
RA groups. Thus, 22.3 ⫾ 3.5% (n ⫽ 6) and 23.4 ⫾ 3.1%
(n ⫽ 4) of NK cells from the SpA and RA groups,
respectively, expressed HLA–DR when cocultured with
FLS, as compared with 14.9 ⫾ 2.3% (n ⫽ 4) of NK cells
from the OA group (P ⬍ 0.05 for both comparisons)
(Figure 2B).
Given that IL-6 was one of the most abundant
cytokines produced by FLS under our experimental
conditions (see Table 2), we wished to determine
712
CHAN ET AL
Figure 3. Fibroblast-like synoviocyte (FLS) promotion of contact-dependent natural killer (NK)
cell production of interferon-␥ (IFN␥) and proliferation, as determined by fluorescence-activated
cell sorter analysis. A, Permeabilized CD3–,CD56⫹ NK cells showing IFN␥ secretion versus CD56
expression in NK cells cultured in media only, with autologous FLS, with FLS in the presence of
a Transwell after 24 hours, or with phorbol myristate acetate (PMA)/ionomycin (ION) (positive
control) after 6 hours. Results are from a representative patient of 6 spondylarthritis (SpA), 4
rheumatoid arthritis (RA), and 8 osteoarthritis (OA) patients evaluated. Values are the percentage
of cells in each compartment. B, Intensity of 5,6-carboxyfluorescein diacetate N-succinimidyl ester
(CFSE-DA) labeling of NK cells (shaded histograms) cultured with media only, with FLS, with FLS
in the presence of a Transwell, or with 100 IU/ml of interleukin-2 (IL-2; positive control) after 7
days. Open histograms show the autofluorescence of unlabeled cultured NK cells. Arrow shows
major cell populations that have divided once. Results are representative of 3 experiments using
cells from 6 SpA, 4 RA, and 8 OA patients. Values are the percentage of cells that have undergone
division.
MUTUAL STIMULATION OF FLS AND NK CELLS IN INFLAMMATORY ARTHRITIS
713
Figure 4. Natural killer (NK) cell survival promoted by fibroblast-like synoviocytes (FLS) and
up-regulation of interleukin-15 (IL-15) expression on FLS by coculture with NK cells, as
determined by fluorescence-activated cell sorter analysis. A, CD3–,CD56⫹ NK cells showing
annexin V staining versus 7-aminoactinomycin D (7-AAD) staining of NK cells cultured for 7 days
in media only, with IL-15–blocking monoclonal antibody (mAb), with autologous FLS, with FLS in
the presence of IL-15–blocking mAb, or with FLS in the presence of isotype control antibody (Ab).
Values are the percentage of cells in each compartment. B, FLS showing cell surface IL-15
expression (shaded histograms) after culture for 7 days in media only or with NK cells. Open
histograms show isotype control IgG1 antibody (background staining). Cells were first gated on
CD56–, NKp30–, and NKp46 mAb. Results are representative of 3 experiments using cells from 6
spondylarthritis (SpA), 4 rheumatoid arthritis (RA), and 8 osteoarthritis (OA) patients.
whether IL-6 was involved in the activation of NK cells
in contact with FLS. Addition of saturating concentrations of neutralizing mAb against IL-6 to the NK
cell/FLS coculture did not reduce the expression of
CD69 or HLA–DR on NK cells.
FLS stimulation of NK cell IFN␥ production and
proliferation. We next sought to establish whether coculture of NK cells with FLS is associated with increased
NK cell effector function. IFN␥ secretion from NK cells
was measured by intracellular cytokine staining and
FACS analysis. Figure 3A shows the results of a representative experiment of 3 that were performed for each
patient. NK cells derived from patients in each of the
arthritis groups did not constitutively secrete significant
quantities of IFN␥ when cultured in media alone (overall mean ⬍1%; mean ⫾ SD in SpA patients, 0.9 ⫾ 0.3%,
714
in RA patients, 0.9 ⫾ 0.3%, and in OA patients, 0.5 ⫾
0.2%). In coculture with FLS, intracellular IFN␥ was
increased after 24 hours as compared with baseline. This
was higher in SpA (14.6 ⫾ 2.3%) and RA (14.0 ⫾ 1.5%)
patients than in OA patients (4.2 ⫾ 3.0%). Cocultures of
NK cells and FLS separated by a 0.4-␮m Transwell did
not result in a significant increase in NK cell secretion of
IFN␥ (2.3 ⫾ 0.8% in SpA, 2.0 ⫾ 0.3% in RA, and 1.8 ⫾
0.8% in OA), showing that NK cell/FLS contact is
important in this interaction.
Figure 3B shows that a proportion of CFSE-DA–
labeled NK cells from SpA and RA patients divide upon
culture with FLS. Over 20% of SpA and RA NK cells
cultured with FLS underwent cell division, as compared
with 5% of OA NK cells (Figure 3B). There was also a
greater than 10-fold rise in the percentage of SpA and
NK cells undergoing cell division when cultured with
FLS as compared with media alone. The number of SpA
and RA NK cells undergoing proliferation was reduced,
but not abolished, when contact with FLS was lost
through the presence of a Transwell.
Promotion of NK cell survival by SpA and RA
FLS and dependence upon IL-15. We next sought to
establish whether the survival of NK cells is promoted by
FLS. NK cell survival was promoted at a higher level in
SpA and RA patients as compared with OA patients. A
representative experiment from 6 SpA, 4 RA, and 8 OA
samples is shown in Figure 4A. In all 3 forms of arthritis,
NK cells cultured in media alone underwent high levels
of apoptosis (mean ⫾ SD 33.7 ⫾ 3.6% [n ⫽ 6] in SpA,
31.5 ⫾ 4.4% [n ⫽ 4] in RA, and 36.7 ⫾ 3.4% [n ⫽ 8] in
OA), and this was further increased in the presence of
blocking anti–IL-15 mAb (68.7 ⫾ 2.7% in SpA, 68.2 ⫾
1.5% in RA, and 71.1 ⫾ 3.6% in OA). In OA patients,
19.1 ⫾ 2.7% of NK cells cultured with FLS were
undergoing apoptosis (annexin V⫹, 7-AAD⫹), as compared with 5.9 ⫾ 1.3% in SpA patients and 6.5 ⫾ 1.3%
in RA patients (P ⬍ 0.01 for both comparisons).
We wished to determine if IL-15 was implicated
in the promotion of NK cell survival by FLS. We found
that survival of NK cells was significantly inhibited by a
combination of blocking anti–IL-15 mAb and a polyclonal goat IgG anti–IL-15R␣ (22.5 ⫾ 3.7% in SpA,
22.3 ⫾ 5.8% in RA, and 28.5 ⫾ 5.5% in OA; P not
significant for SpA or RA versus OA), but not by an
irrelevant isotype–matched mAb (6.2 ⫾ 1.0% in SpA,
7.0 ⫾ 2.4% in RA, and 15.3 ⫾ 2.2% in OA; P ⬍ 0.01 for
SpA or RA versus OA).
Up-regulation of cell surface IL-15 expression
upon contact of FLS with NK cells. The expression of
IL-15 on the surface of FLS was determined by FACS
CHAN ET AL
analysis of nonpermeabilized cells (Figure 4B). At rest,
the surface expression of IL-15 on FLS was higher in
SpA patients (mean ⫾ SD 6.8 ⫾ 1.8% [n ⫽ 6]) and RA
patients (8.9 ⫾ 1.5% [n ⫽ 4]) as compared with that in
OA patients (2.3 ⫾ 0.9% [n ⫽ 8]) (P ⬍ 0.005 for both
comparisons). Surface IL-15 expression increased with
time upon coculture with NK cells. Thus, on day 7,
surface expression of IL-15 by FLS was 16.9 ⫾ 2.2% in
SpA patients, 20.9 ⫾ 3.3% in RA patients, and 4.1 ⫾
1.4% in OA patients (P ⬍ 0.001 for SpA or RA versus
OA).
Production of a proinflammatory cytokine milieu
following interactions between RA and SpA FLS with
NK cells. We next determined the cytokine profile
resulting from interactions between NK cells and FLS. A
total of 18 cytokines and 9 stromal factors were measured in culture supernatants using the multiplex assay.
Supernatants obtained on day 7 from cultures of NK
cells or FLS alone or from cocultures of NK cells and
FLS were analyzed (Table 2). NK cells from all 3
arthritis groups cultured alone produced low but detectable amounts of vascular endothelial growth factor
(VEGF), RANTES, and MMP-9. Additionally, SpA NK
cells produced IL-15, and both SpA and RA NK cells
produced low levels of IL-1␤, IFN␥, and MIP-1␤. FLS
cultured alone produced significant quantities of IL-6
(greatest in SpA patients), GM-CSF, and MCP-1, as well
as low levels of IL-1␤ and IL-8.
NK cell/FLS coculture resulted in the production
of high levels of IL-6, IL-8, GM-CSF, and MCP-1. In
addition, small but significant levels of TNF␣, IL-5,
IL-15, granulocyte colony-stimulating factor (G-CSF),
and IFN␥-inducible 10-kd protein were now produced. For example, TNF␣ levels were increased in all
NK cell/FLS coculture supernatants. Thus, the
mean ⫾ SD TNF␣ levels in supernatants from cocultures of NK cells and FLS were 7.6 ⫾ 0.3 pg/ml in SpA
and 8.8 ⫾ 1.2 pg/ml in RA. In OA, the TNF␣ levels in
NK cell/FLS supernatants were significantly lower at
4.1 ⫾ 1.0 pg/ml (P ⬍ 0.001 versus SpA and versus RA).
Up-regulation of MMP-3 in cocultures of NK
cells and FLS from SpA patients. In the stromal factor
panel, the level of MMP-3 was very substantially increased in cocultures of NK cells and FLS from patients
with SpA (Table 2). Levels were higher than those in the
RA and OA NK cell/FLS cocultures, and were ⬃1,000
times higher than the level produced by cultures of FLS
alone. In addition, coculture produced increased levels
of MMP-9 and detectable levels of MMP-13.
MUTUAL STIMULATION OF FLS AND NK CELLS IN INFLAMMATORY ARTHRITIS
DISCUSSION
Fibroblast-like synoviocytes have previously been
shown to play an important role in RA through T cell
activation as well as the promotion of both T lymphocyte
and neutrophil survival (26). This study is the first to
show that FLS from patients with arthritis interact with
syngeneic NK cells resulting in mutual stimulation and
the release of proinflammatory cytokines. NK cells
physically interact with FLS, undergoing pseudoemperipolesis, activation, IFN␥ production, proliferation, and
increased survival, in addition to maturation, as described recently (27). Our data do not prove that NK
cell–FLS contact is essential for these effects. However,
in addition to observing their direct interaction, we
found that their separation in Transwell cultures prevented NK cell IFN␥ production and proliferation,
showing that at least close proximity is required. We also
demonstrated increased surface expression of IL-15 by
FLS upon coculture with NK cells, suggesting one
possible mechanism, although further detailed studies of
the mechanics will be required. All of these features
were significantly enhanced in both RA and SpA cells as
compared with OA cells, implying a preexisting or
induced functional enhancement of either FLS or peripheral blood NK cells (or both) in these diseases.
We demonstrated not only that basal levels of
surface expression of IL-15 are higher in SpA and RA
FLS than in OA FLS, but that the interaction with NK
cells results in further increases in surface IL-15 expression on FLS. We further provide evidence that IL-15
promotes increased NK cell survival, since the presence
of IL-15–blocking antibody resulted in increased apoptosis. However, the survival of NK cells promoted by
FLS is not completely IL-15–dependent, since IL-15
blockade in NK cell/FLS cocultures resulted in a lower
level of NK cell apoptosis as compared with IL-15
blockade in NK cells cultured alone. Previous studies
have shown that IL-15 is crucial in NK cell survival
(32,33). This increased expression of IL-15 on FLS may
also act in an autocrine manner to promote FLS activation, proliferation, and resistance to apoptosis (34).
Thus, our data support an important role of IL-15 in NK
cell–FLS interactions in inflammatory arthritis but not in
OA. Our data also suggest that strategies aimed at
blocking this interaction (e.g., with anti–IL-15 mAb) are
likely to be effective in reducing both inflammation and
joint damage.
The finding of increased NK survival induced by
FLS further extends the role of fibroblasts in supporting
the survival of leukocytes, which has already been shown
715
for T lymphocytes (9), neutrophils, dendritic cells, B
lymphocytes, and plasma cells (26). Maintaining the
inappropriate survival of leukocyte subpopulations is
likely to be an important contribution by FLS to the
persistence of chronic inflammatory diseases.
The interaction of NK cells and FLS also results
in NK cell activation, proliferation, and production of a
proinflammatory milieu consisting of high levels of IL-6,
IL-8, GM-CSF, MCP-1, and MMP-3. Production was
5–10 times higher in RA and SpA patient-derived samples than in OA patient-derived samples, except for
IL-6. Increased production of IL-15, IFN␥, and VEGF
was also seen in cells from SpA and RA patients, albeit
at slightly lower levels. IL-6 was abundantly produced by
FLS and is likely to be important in NK stimulation
(35–37). However, we were unable to demonstrate
blockade of NK cell activation with anti–IL-6 mAb,
perhaps because the action of IL-6 on NK cell activation
is mediated by the production of IL-2 by T cells (36).
The high levels of IL-8 production observed in NK
cell/FLS cocultures were most marked in SpA and RA
and could play a significant role in attracting neutrophils
into the joints in all 3 diseases.
Interestingly, we also showed the ability of NK
cell/FLS cocultures to produce large amounts of MCP-1.
This was consistently greater in cocultures of NK cells
and FLS from RA and SpA patients as compared with
cells from OA patients. MCP-1, GM-CSF, and TNF␣
are involved in monocyte/dendritic cell activation and
maturation (38,39). Therefore, our data suggest that NK
cells and FLS could be involved in a cumulative and/or
synergistic effect with monocytes in the promotion of
inflammation. Of note, the production of type I IFNs
(IFN␣ and IFN␤) by FLS was not determined in our
study, but has previously been shown to be important in
the survival of T cells in RA patients (10).
Although our data were obtained in vitro and we
used peripheral blood NK cells, our findings are consistent with the previously reported finding of activated NK
cells within the joints of patients with inflammatory
arthritis (38). In the future, our data should be confirmed using synovial fluid NK cells. Further study of NK
receptor–ligand interactions, such as natural killer cell
receptor group 2D/major histocompatibility complex
class I chain–related molecule (40), KIR/HLA (19), and
lymphocyte function⫺associated antigen 1/intercellular
adhesion molecule 1, between NK cells and FLS, respectively, also further elucidate the mechanisms underlying
the proinflammatory response seen in our study.
Overall, our data suggest that similar modes and
levels of immune activation occur following NK interac-
716
CHAN ET AL
tions with FLS in RA and SpA patients. Although IL-13,
IFN␥, G-CSF, RANTES, and MMP-3 production were
all increased most markedly in SpA and more so in RA
than in OA, it is uncertain, given the small sample size,
whether these differences could be of clinical significance. It is more likely that the NK cell–FLS interaction
amplifies and/or prevents resolution of the inflammatory
processes in both SpA and RA. Our results also show
differential production of MMP-3 by activated FLS,
correlating with the elevated serum and synovial fluid
levels of MMP-3 in patients with ankylosing spondylitis
(41,42).
In summary, our experiments show that in both
RA and SpA patients, the interaction of FLS with NK
cells results in enhanced NK cell survival and in the
elaboration of multiple proinflammatory and chemotactic products, which may contribute to the persistence of
inflammation.
9.
10.
11.
12.
13.
14.
15.
AUTHOR CONTRIBUTIONS
Drs. Chan and Bowness had full access to all of the data in the
study and take responsibility for the integrity of the data and the
accuracy of the data analysis.
Study design. Chan, Kollnberger, Buckley, Bowness.
Acquisition of data. Chan, Filer, Parsonage, Gundle.
Analysis and interpretation of data. Chan, Filer, Kollnberger, Buckley,
Bowness.
Manuscript preparation. Chan, Buckley, Bowness.
Statistical analysis. Chan.
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