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2,*
*
Division of Rheumatology and
Department of Orthopedic Surgery, University Hospital, Geneva, Switzerland;
Department of Rheumatology, University Hospital, Nij-megen, The Netherlands; and
§
Department of Pathology, University Medical Center, Geneva, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which play a crucial role in cartilage degradation through
their inducing effects on matrix metalloproteinases. IL-4 is able to
suppress detrimental effects of IL-1 and TNF- by stimulating IL-1Ra
production (1), and down-regulating TNF receptor
(2), transcription of metalloproteinases (3),
and synoviocyte proliferation (4). These effects could be
exploited in the treatment of RA and/or osteoarthritis (OA). In vivo,
however, preliminary human studies in RA patients proved disappointing.
Interestingly, studies on animal models of RA showed that IL-4
treatment protects cartilage destruction, but does not reduce synovial
expansion and inflammation (5, 6, 7, 8, 9). Furthermore, synovial
hyperplasia is even enhanced after IL-4 treatment (7, 9).
Synovial hyperplasia is thought to be due, at least in part, to
inadequate apoptosis (10). The synovial expansion induced
by local IL-4 treatment might well be due to an antiapoptotic effect on
synoviocytes because IL-4, through its receptor (IL-4R),
prevents apoptosis of different cell types (11, 12). In this work, we tested the ability of IL-4 to protect human synoviocytes or synovial explants from apoptosis induced by the NO donor sodium-nitro-prusside (SNP). NO was chosen as a proapoptotic agent due to its overproduction in RA joint, and its ability to induce apoptosis of human synoviocytes in vitro (10, 13).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synovial membranes were obtained from OA and RA patients during joint replacement. Normal synovial cells were obtained during joint replacement for hip fracture. Isolated cells (passages 2–4), or synovial explants of 7–8 mm3 were used in experiments. For cell isolation, synovium was finely cut and digested with collagenase, as previously described (14). Isolated cells were maintained in Dulbecco’s medium supplemented with 10% FCS (Life Technologies, Grand Island, NY). Thirteen OA patients were receiving only nonsteroidal antiinflammatory drugs and/or acetaminophen. Three patients with RA were receiving Methotrexate (one patient) or Methotrexate plus Hydroxychloroquine (two patients). Results obtained with RA synoviocytes, however, did not appear to be different from those obtained with OA synoviocytes.
Apoptosis induction
The NO donor, SNP, was chosen as a proapoptotic agent because of its ability to induce apoptosis in synoviocytes (10, 13). Synoviocytes or synovial explants were cultured in Dulbecco’s medium supplemented with 1–5% FCS. After preincubation with IL-4 (Genzyme, Cambridge, MA, and R&D Systems, Minneapolis, MN), IL-10, or IL-13 (PeproTech, Rocky Hill, NJ) for 48 h, cells or tissues were treated for an additional 24 h with SNP (Sigma-Aldrich, St. Louis, MO). In some experiments, wortmannin (Sigma-Aldrich), LY 294002, bisindolylmaleimide I, Gö 6983, Gö 6976 (Calbiochem, La Jolla, Ca), and insulin-like growth factor (IGF)-1 (R&D Systems) were added alone or concomitantly with ILs, during pretreatment.
Microscopic quantification of apoptosis
A total of 104 synoviocytes in 0.4 ml of medium were seeded in eight-well chamber slides (Nalge Nunc International, Naperville, IL). Cells were fixed 15 min in 3.7% paraformaldehyde, and washed with PBS. Staining of the cells was done with 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide for 15 min, simultaneously. After washing of the cells with PBS, cover slips were mounted on chamber slides by Fluoprep (Bio Mérieux SA, Marcy l’Etoile, France), and cells were observed with a fluorescence microscope. Apoptotic cells were readily recognized, as they had a condensed or fragmented nucleus. Percentage of nonapoptotic cells was calculated as the average number from duplicate cell cultures, each counted from five microscope fields (60–80 cells), and expressed as percentage in relation to the number of control cells (100%).
Apoptosis detection by annexin V-FITC
Phosphatidylserine (PS) residues on cell surface were detected by annexin-V binding, as an index of apoptosis (15, 16). Cells exposed to IL-4 for 48 h and treated with SNP for an additional 2 or 24 h were removed from culture dishes with trypsin/EDTA. After a rinsing step with PBS containing 2% of BSA, PS were detected by labeling with annexin V-FITC (0.5 µg/ml) according to the manufacturer’s instructions (TACS annexin V-FITC apoptosis detection kit; R&D Systems). Cells were then analyzed by flow cytometry on a FACScan (Becton Dickinson, Mountain View, CA). Results were integrated with the CellQuest software (Becton Dickinson) and expressed as percentage of annexin V-positive cells in typical histogram profiles. U937 monocytic cell line was used as a positive control for apoptotic cell death. Apoptosis in U937 was induced with the proapoptotic agent, camptothecin (Sigma-Aldrich), used at 10 µg/ml for 4 h.
3-(4,5-Dimethylthiazol-2-gl)-5-(3-carboxymethoxylphenyl)-2-(4-sulphophenyl-2Htetrazolium inner salt (MTS) survival assay
Cell survival was measured as mitochondrial NADH/NADPH-dependent dehydrogenase activity, resulting in the cellular conversion of the tetrazolium salt MTS into a soluble formazan dye (17). Briefly, cells were seeded in 96-well plate at the concentration of 5000/200 µl well. For induction of Fas-mediated apoptosis, 4 x 104 cells/200 µl of medium were used. After treatments, 40 µl of MTS solution (Promega, Madison, WI) was added in each well for 2–3 h. Finally, colorimetric measurement of formazan dye was performed on a spectrophotometer with an OD reading at 490 nm (17).
DNA end labeling
Total DNA of human primary synoviocytes was isolated and labeled
with Laddering-isotopic kit from R&D Systems, using Klenow enzyme and
the [-32P]dCTP or
[-32P]dATP (Hartmann Analytic, Braunschweig,
Germany). One microgram of labeled DNA was electrophoresed on
0.6%–1% Trevis agarose gel in 1x Tris acetate EDTA. Gels were fixed
in 10% acetic acid for 1 h at 4°C, vacuum dried, and exposed
either to x-ray films for 10–30 min and/or a phosphor imaging
cassette. Intensity of signals was calculated by densitometry, using
Molecular Analyst software from Bio-Rad (Hercules, CA), or by a model
425 PhosphorImager (Molecular Dynamics, Sunnyvale, CA), using
ImageQuant software. Relative percentage of apoptosis was calculated in
relation to the value obtained by SNP treatment (100%). DNA from
synovial explants was extracted by phenol, after 24-h digestion of
minced frozen tissue with 100 mM NaCl, 10 mM Tris-Cl, pH 8, 25 mM EDTA,
pH 8, 0.5% SDS, and 0.2 mg/ml proteinase K (Boehringer Mannheim,
Indianapolis, IN) at 50°C. End labeling of DNA and electrophoresis
were done as explained above.
Pulse field gel electrophoresis (PFGE)
Synoviocytes were harvested by trypsinization, washed with PBS,
and embedded in 1% low melting agarose in lysis buffer (20 mM NaCl,
100 mM EDTA, 10 mM Tris, pH 7.5). Solidified agarose plugs were
incubated overnight in lysis buffer supplemented with 1% sarcosyl and
1 mg/ml of proteinase K, at 50°C. Plugs were dialyzed for 48 h
in 50 mM EDTA, 10 mM Tris, pH 8, at 4°C. Gels (1.5%) prepared in
0.5x TBE, using the pulse field certificated agarose (Bio-Rad), were
loaded with agarose plug pieces, each containing 0.5 x
106 cells. Electrophoresis was conducted in
prechilled (10°C) 0.5x Tris borate EDTA buffer, at 200 V. The switch
time was ramped from 50 to 90 s for 21 h. DNA size standard
(0.05–1 Mb) was Lambda Ladder (Bio-Rad). Gels were stained in 0.5x
Tris borate EDTA buffer with ethidium bromide (1.5 µg/ml) for 30 min
at 4°C, and photographed. Gels were depurinated in 0.25 M HCl for 30
min, and then denaturated in 0.5 M NaOH, 1.5 M NaCl. Following
neutralization in 0.5 M Tris, 1.5 M NaCl, pH 7, the transfer of DNA to
-Probe GT nylon membrane (Bio-Rad) was done overnight through
capillary action using 10x SSC. Blotted membranes were exposed to
80°C for 30 min. Hybridization was done at 65°C for 24 h in
0.25 M sodium phosphate, pH 7.2, containing 7% SDS with addition of
[-32P]dCTP-labeled Cot-1 human DNA
(Boehringer Mannheim), enriched in repetitive sequences. Membranes were
washed twice for 15 min in 20 mM sodium phosphate, pH 7.2, 1% SDS at
65°C, and then exposed to x-ray films for 24–72 h.
Statistics
Values of p were obtained using the Mann-Whitney test and considered significant when lower than 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis of human synoviocytes was induced by the NO donor SNP.
Following exposure to 2 mM SNP for 24 h, no cells survived (Fig. 1
) and morphology of the cells showed a
chromatin condensation characteristic of apoptosis (Fig. 1C). However, when cells were pretreated by IL-4, 65–86%
of them were protected, based on microscopic quantification of
nonapoptotic cells (Fig. 1, D and E). To confirm
the proapoptotic effects of SNP and the protective effects of IL-4,
apoptosis was also evaluated by measurement of PS exposure with annexin
V-FITC (Fig. 2). Likewise, SNP induced a
strong increase of annexin V-positive apoptotic cells (20% at
2 h and more than 60% at 24 h), which was nearly
completely blocked by IL-4. Comparable degrees of IL-4-mediated
protection from SNP-induced apoptosis were obtained when cell survival
was measured by MTS colorimetric assay (Fig. 3, A and B).
Dose-response analyses performed by both assays showed that the lowest
concentrations inducing complete apoptosis of human synovial cells,
within 24 h, usually ranged between 1 and 2 mM, and that
antiapoptotic effect of IL-4 was similar for OA (Figs. 1E
and 3A), RA (Figs. 1E and 3B), and
normal synoviocytes (data not shown). These results also suggest that
medications used in RA do not influence the effects of IL-4.
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). The DNA from SNP-treated cells was
fragmented into high m.w. (HMW) fragments (Fig. 4A,
line 2). Such HMW DNA fragments, usually ranging from 30 kb
to 1 mbp, found in all tested apoptotic cells, are considered an
essential sign of apoptosis (18, 19, 20, 21). Accumulation of 30-
to 50-kb DNA fragments is a characteristic pattern of detached
apoptotic cells, especially in cell lines in which DNA laddering does
not occur (20). DNA from SNP-treated synoviocytes indeed
showed an accumulation of fragments around 50 kb (Fig. 4A,
line 2). Pretreatment of synoviocytes with IL-4 before SNP
exposure prevented or dramatically diminished DNA fragmentation (Fig. 4A, line 3).
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The effects of IL-13 and IL-10, two other Th2 cytokines, were
analyzed in additional experiments. IL-13 and IL-4, due to shared
receptors, are known to overlap in several biological functions.
Indeed, the effects of high doses of IL-13 were comparable with those
of IL-4 (Fig. 5). However, at lower
concentrations (5 and 0.5 ng/ml), IL-13 protection was lower compared
with that obtained with IL-4 (26% vs 67%, and 9% vs 50%,
respectively). In contrast, IL-10 did not show any protective effect
against NO-induced apoptosis, even at concentrations as high as 50
ng/ml.
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To further document the effects of IL-4 and IL-13, experiments
were also performed on synovial explants, obtained during joint
replacement. Explants were cultured for 2 days in the presence or
absence of IL-4 or IL-13, followed by 24-h SNP treatment.
Apoptosis was analyzed and quantified by
32P Klenow end labeling of total DNA separated on
conventional agarose gel. We have developed this simple apoptosis test
after having observed, in isolated human synoviocytes, a correlation
between apoptosis and intensity of Klenow end-labeled HMW DNA
fractions, able to migrate into agarose gel (Fig. 6A). Radioactive labeling of
total DNA by Klenow revealed two bands marked as "HMW DNA" and
"23 kb" correlating with the degree of apoptosis (Fig. 6A). Analysis of both signal intensities by phosphor imager
showed that correlation with the level of apoptosis was better for the
HMW DNA (Fig. 6B), which was therefore used for
quantification in subsequent experiments. Using this method, the
densitometric analysis of HMW DNA (Fig. 6C) from synovial
explants treated with IL-4 or IL-13 before SNP exposure indicated that
protective effects of IL-4 and IL-13 were 88% and 33%, respectively
(Fig. 6D).
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Because IL-4 was shown to exhibit its antiapoptotic effect, at
least in part through the insulin receptor substrate (IRS) pathway
(11), the effect of the phoshatidylinositol-3 kinase
(PI-3-K) inhibitors wortmannin and LY294002 was tested by concomitant
addition during IL pretreatment. LY294002 markedly decreased IL-4 and
IL-13 protective effects (Fig. 7, A and B, respectively). Similar effects for both
cytokines were observed for wortmannin, at the concentrations 10–1000
nM (data not shown). Furthermore, addition of IGF-1, which uses the IRS
pathway, significantly, increased cell survival during IL-4 or IL-13
pretreatment (p = 0.0163 and p
= 0.009, respectively) (Fig. 8). These
results contribute to indicate that, in human synoviocytes, as
previously shown for other cell types (11), antiapoptotic
effect of IL-4 involves the IRS pathway.
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PKCs represent one of the potential IRS-dependent or independent
pathways of IL-4 antiapoptotic signaling. We therefore tested the
effects of different PKC inhibitors on the protective effects of IL-4.
We used Gö6976 which inhibits PKC , 
1, µ, Gö6983
which inhibits PKC , , 
, 
, and bisindolylmaleimide I which
inhibits PKC , 1, , 
. Gö 6976 significantly
diminished the antiapoptotic effect of IL-4 (p
= 0.049) (Fig. 9), while Gö 6983
and bisindolylmaleimide I had no significant effects. Taken together,
these differential effects of the PKC inhibitors suggest an involvement
of PKCµ in the antiapoptotic effects of IL-4. Interestingly,
pretreatment of synoviocytes with Gö 6983
(p = 0.049) and, to a lesser extent,
bisindolylmaleimide I (not significant) had a consistent protective
effect against SNP apoptosis, which suggests the involvement of PKC.
None of the inhibitors used in these experiments affected the viability
of the cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-4, which opposes several effects of IL-1 and TNF- on
synoviocytes, is contemplated for the treatment of RA and/or OA.
However, the fact that beneficial effects of IL-4 on cartilage
destruction in animal models of RA are not accompanied by suppressive
effects on synovial hyperplasia have led to question its use in gene
therapy (6, 7). Misbalance between synovial proliferation
and low frequency of apoptosis is considered a critical event in RA
(22). Apoptosis of synoviocytes can be induced by NO
donors (13), c-Myc antisense oligodeoxynucleotides
(23), anti-Fas Abs (24), or apoptotic
agents, such as verteporfin (25). Treatment with
bisindolylmaleimide VIII, which facilitates Fas apoptosis, ameliorates
symptoms in Lewis adjuvant arthritis (26). In contrast,
inactivation of the p53 tumor suppressor gene in synoviocytes was shown
to prevent NO-induced apoptosis and to render the cells more invasive
(10). Therefore, proteins having antiapoptotic activities
could have a detrimental effect on disease, by favoring synovial
expansion.
Apart from antiapoptotic effects on isolated synoviocytes, we showed
that IL-4 and IL-13 also protect cultured synovial explants from
NO-induced apoptosis. These results suggest that besides beneficial
effects on cartilage, exogenous IL-4 and IL-13 may promote hyperplasia
of the synovium. In addition, these results indicate that endogenous
IL-13 and, to a lesser extent, IL-4 could be partially responsible for
synovial hyperplasia during pathophysiological processes in RA. IL-13
is consistently found in synovial fluids of patients suffering from RA
(27), and 35% of RA patients have been shown to have
abnormally high serum levels of IL-4 (28). Furthermore,
the antiinflammatory and immunosuppressive effects of glucocorticoids
are thought to be exerted, at least in part, by down-regulation of
IL-4R (29). Human synoviocytes express IL-4R and
IL-13R1, and overexpress IL-13R2, but not the common -chain of
IL-2R (30).
We show in this study that antiapoptotic activities of IL-4 can be
down-regulated by the PI-3-K inhibitor, LY294002, and the selective PKC
inhibitor, Gö6976. IRS-dependent pathway is known to be involved
in the antiapoptotic signaling of IL-4R (11). The
differential results obtained in this study with Gö6983,
Gö6976, and bisindolylmaleimide I suggest that activation of
PKCµ may be responsible for cell protection by IL-4. Recently, PKCµ
expression was also found to be correlated with the proliferative state
of murine keratinocytes and NIH3T3 fibroblasts (31). These
results are also consistent with a recent study showing that inhibition
of PKC with Gö6976 results in a significant increase of both
caspase 3 and caspase 8 activity in anti-Fas-treated Jurkat cells
(32). The partial protection from SNP-induced apoptosis by
Gö6983 alone suggests the involvement of PKC. A recent report
showed that rottlerin, a PKC inhibitor, prevented anti-Fas
apoptosis in Jurkat cells (33). Preliminary results from
our laboratory showed that SNP causes a modest but consistent induction
of caspase 3 in synoviocytes, but not in IL-4-pretreated cells.
Experiments are in progress to clarify the role of PKCs in caspase
activation. In the light of our results and those of others, possible
pathways implicated in IL-4/13 antiapoptotic effects are summarized in
Fig. 10.
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functions will tell whether
antiapoptotic effects can be uncoupled from other effects of IL-4
and/or IL-13, which may be useful for new therapeutic approaches in RA
and OA.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Rheumatology, University Hospital Sart-Tilman, Liège, Belgium.
3 Address correspondence and reprint requests to Dr. Pierre-André Guerne, Division of Rheumatology, University Hospital, 26 Avenue Beau Séjour, 1211 Geneva 14, Switzerland.
4 Abbreviations used in this paper: RA, rheumatoid arthritis; HMW, high m.w.; IGF, insulin-like growth factor; IRS, insulin receptor substrate; MTS, 3-(4,5-dimethylthiazol-2-gl)-5-(3-carboxymethoxylphenyl)-2-(4-sulphophenyl-2H tetrazolium inner salt; OA, osteoarthritis; PFGE, pulse field gel electrophoresis; PI-3-K, phoshatidylinositol-3 kinase; PKC, protein kinase C; PS, phosphatidylserine; SNP, sodium-nitro-prusside.
Received for publication January 18, 2000. Accepted for publication December 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-4. J. Immunol. 152:2060.[Abstract]
’s effect on rheumatoid synovial fibroblasts. Rheumatol. Int. 14:21.[Medline]
RIII and acquire annexin V binding sites during apoptosis in vitro. Blood 85:532.. J. Biol. Chem. 275:23065.This article has been cited by other articles:
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  K. Silbermann, G. Schneider, and R. Grassmann Stimulation of interleukin-13 expression by human T-cell leukemia virus type 1 oncoprotein Tax via a dually active promoter element responsive to NF-{kappa}B and NFAT J. Gen. Virol., November 1, 2008; 89(11): 2788 - 2798. [Abstract] [Full Text] [PDF]
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 L. H. Wei, Y. Yang, G. Wu, and L. J. Ignarro IL-4 and IL-13 upregulate ornithine decarboxylase expression by PI3K and MAP kinase pathways in vascular smooth muscle cells Am J Physiol Cell Physiol, May 1, 2008; 294(5): C1198 - C1205. [Abstract] [Full Text] [PDF]
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 W.-U. Kim, S. S. Kang, S.-A. Yoo, K.-H. Hong, D.-G. Bae, M.-S. Lee, S. W. Hong, C.-B. Chae, and C.-S. Cho Interaction of Vascular Endothelial Growth Factor 165 with Neuropilin-1 Protects Rheumatoid Synoviocytes from Apoptotic Death by Regulating Bcl-2 Expression and Bax Translocation J. Immunol., October 15, 2006; 177(8): 5727 - 5735. [Abstract] [Full Text] [PDF]
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B. Uzan, H.-K. Ea, J.-M. Launay, J.-M. Garel, R. Champy, M. Cressent, and F. Liote A Critical Role for Adrenomedullin-Calcitonin Receptor-Like Receptor in Regulating Rheumatoid Fibroblast-Like Synoviocyte Apoptosis J. Immunol., May 1, 2006; 176(9): 5548 - 5558. [Abstract] [Full Text] [PDF]
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 Y. Fujitsu, K. Fukuda, K. Kimura, K. Seki, N. Kumagai, and T. Nishida Protection of Human Conjunctival Fibroblasts from NO-Induced Apoptosis by Interleukin-4 or Interleukin-13 Invest. Ophthalmol. Vis. Sci., March 1, 2005; 46(3): 797 - 802. [Abstract] [Full Text] [PDF]
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M. W. Wynes and D. W. H. Riches Induction of Macrophage Insulin-Like Growth Factor-I Expression by the Th2 Cytokines IL-4 and IL-13 J. Immunol., October 1, 2003; 171(7): 3550 - 3559. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
DNA;
line 1, control; line 2, 0.25 mM SNP;
line 3, 0.5 mM SNP; line 4, 1 mM SNP;
line 5, 1.5 mM SNP; line 6, 2 mM SNP. HMW
DNA is shown by the arrow. B, Signal density values of
labeled HMW DNA, shown in A, scanned by phosphor imager,
and expressed in arbitrary units. The figures are representative
of two experiments. C, Radiography of dried gel,
showing the region of HMW DNA from human synovial explants. Line
1, Control; line 2, IL-4 + SNP; line
3, IL-13 + SNP; line 4, SNP. B,
Signal density values of HMW DNA shown in C scanned by
phosphor imager and expressed in arbitrary units. The experiment was
performed three times (two with synovial explants from OA patients
(presented) and one with a normal synovial explant, with consistent
results).
