Paquet et al.
TNF gene targeting via TFO and arthritis
page # 1
Supporting Information: Material and Methods
Isolation and cultivation of rat chondrocytes and synoviocytes
Synoviocytes were isolated from synovial membranes of healthy Wistar male rats (6 weeks old,
150 to 175 g) (Charles River, Saint-Aubinles-Elbeuf, France), digested overnight by a mix of
collagenase and dispase (Roche) (at 0.1 units/mL and 0.8 units/mL respectively). Chondrocytes
were isolated from femoral heads by sequential digestion with Pronase (2 mg/mL, 2 h) and
collagenase B (1.5 mg/mL, overnight) (Roche, Meyland, France) as previously described [1].
Chondrocytes were used in the first and synoviocytes in the third passage to prevent
dedifferentiation.
Transient transfections
Chondrocytes and synoviocytes were seeded on 24-well culture plates at 60,000 cells per well
and grown to 70% confluence. Cells were transfected with anti-TNF alpha oligonucleotides
(ranging from 1 fM to 100 nM in the final culture medium). Tranfections were performed for two
hours with polyethylenimine reagent (Euromedex, Souffelweyersheim, France) for TFO and with
JetSITM-ENDO (Polyplus-transfection, Illkirch, France) for siRNA, according to the
manufacturers’ recommendations. At 24 hours after transfection, cells were stimulated either with
recombinant rat IL-1β (10 ng/mL, R&D Systems, Lille, France) or with LPS (1 µg/mL) for four
hours (mRNA expression analysis) and 18 hours (production of mediators in supernatants). Cells
were then frozen and stored at -70 °C until processing.
Scoring of arthritis
The disease course was monitored by the repeated assessment of knee joint width using a caliper
(Kroeplin Längenmesstechnik, Schlüchtern, Germany). Raw data were used to estimate joint
circumference using the geometric formula of ellipse circumference:
2π X
(a= knee height and B= knee breadth).
The results were representative of joint swelling and expressed in millimeters as the difference
between the joint size at a given day compared to the size measured just before arthritis induction
(Day zero). Body-weight gain was also monitored daily.
Paquet et al.
TNF gene targeting via TFO and arthritis
page # 2
Weight-bearing assessment protocol
Hindlimb weight bearing was determined using an incapacitance tester (Linton Instrumentation,
Norfolk, UK) consisting of a dual channel weight averager. Weight distribution was measured
between sensitized (intra-articular injection of mycobacterium walls and mBSA) and
contralateral (saline injected) hindlimbs and used as an index of joint discomfort in the arthritic
knee. Rats were carefully placed in an angled Plexiglas chamber positioned so that each hindpaw
rested on a separate force plate. Care was taken to ensure that the animal weight was directed
onto the force plates and not dissipated through the walls of the chamber. The force exerted by
each hindlimb (measured in grams) was averaged over a period of 5 s. Each data point is the
mean of three readings. The percentage of weight distributed onto the sensitized (arthritic)
hindlimb was calculated by the following equation: [weight on ipsilateral hindlimb /(weight on
ipsilateral + weight on contralateral)] x100 [2]. Weight distribution of arthritic rats was compared
to that of control rats (injected bilaterally with saline into the knee joints).
Assessment of proteoglycan metabolism in patellar cartilage
Proteoglycan synthesis was studied by ex vivo incorporation of Na2
35
SO4 into patellar cartilage
as previously described [3]. Data are expressed as the percentage of variation from healthy
controls, with a negative value representing a decrease in proteoglycan synthesis.
Histological analyses
Knee joints were collected at necropsy, fixed immediately for 24 hours in 4% paraformaldehyde,
then decalcified in rapid bone decalcifier (RDO; Apex Engineering, Plainfield, IL, USA) at room
temperature for 6 hours, dehydrated and further fixed in 4% paraformaldehyde before being
embedded in paraffin. Sections (5 μm thick) were rehydrated in a graded ethanol series and
stained with hematoxylin/eosin/saffron, toluidine blue, sirius red and safranin-O. The histological
characteristics of articular cartilage, bone and periarticular soft tissues were scored (adapted from
Mankin et al.) [4] independently by two observers who were blind to the samples. Cartilage
degradation was graded from 0 to 3, where 0 = fully stained cartilage, 1 = loss of proteoglycan
staining in the superficial layer, 2 = complete loss of proteoglycan staining, and 3 = complete loss
of cartilage. The following morphological criteria were used for bone erosion: 0 = normal, 1 =
mild loss of cortical bone at a few sites, 2 = moderate loss of cortical and trabecular bone, and 3 =
marked loss of bone at many sites. Synovium from knee joint was graded using a scoring
Paquet et al.
TNF gene targeting via TFO and arthritis
page # 3
technique adapted from Rooney et al. [5]. Briefly, samples were evaluated on a scale from 0 to 4
(with 0 = normal and 4 = major changes) for hyperplasia of synovial fibroblasts (depth of lining
layer), fibrosis (percentage replacement of loose connective tissue), angiogenesis (number of
proliferating blood vessels), perivascular infiltrates of lymphocytes (percentage of vessels
surrounded by lymphocytes) and tissue infiltration by lymphocytes (size of aggregates,
percentage infiltrating cells).
Assessment of cytokine mRNA expression
Tissue sampling and preparation. Samples of knee joint synovial membrane and patella cartilage
were obtained from rats sacrificed 24 hours after arthritis induction. At necropsy, tissue pieces
were frozen and stored at -80 °C until processing.
RNA extraction and real-time PCR analysis. Frozen tissues (synovial membranes and patella
cartilage) were homogenized immediately after thawing using a dispersing system and mixed
with 350 µL RLT buffer added with β-mercapto-ethanol. Samples were then placed on
QIAshredder columns (Qiagen, RNeasy Kit, Courtaboeuf, France) and mRNA was extracted with
the RNeasy kit® following the manufacturer’s recommendations. A total of 0.5 µg of mRNA
from each sample was then reverse-transcribed with 200 U of Moloney murine leukemia virus
reverse transcriptase (Invitrogen) and oligo(dT) primers (Eurogentec, Liège, Belgium), according
to the supplier’s recommendations. Expression of TNF-α, IL-1β, IL-6, vEGF, iNos and MMP13
was quantified by real-time PCR with the Lightcycler® (Roche) technology and the SYBRgreen
master mix system® (Qiagen, Courtaboeuf, France). For quantification of the changes in gene
expression, the comparative Ct method was used to calculate the relative-fold changes
normalized against the ribosomal RNA [6]. Data are presented as the mean of three samples and
each sample was assayed in triplicate. The gene-specific primer pairs used were as follows:
TNF-α,
IL-1β,
IL-6,
COX2,
MMP13,
sense
antisense
sense
antisense
sense
antisense
sense
antisense
sense
antisense
5'-AGCCCTGGTATGAGCCCATGTA-3',
5'- CCGGACTCCGTGATGTCTAAGT-3';
5'-CTTCCCCAGGACATGCTAGG-3',
5'-CAAAGGCTTCCCCTGGAGAC-3';
5'-CCGGAGAGGAGACTTCACAG-3',
5'-ACAGTGCATCATCGCTGTTC-3';
5’-TACAAGCAGTGGCAAAGGCC-3’,
5’-CAGTATTGAGGAGAACAGATGGG-3’;
5’-AGGCCTTCAGAAAAGCCTTC-3’,
5’-GAGCTGCTTGTCCAGGTTTC-3’;
Paquet et al.
vEGF,
iNos,
S29,
TNF gene targeting via TFO and arthritis
sense
antisense
sense
antisense
sense
Antisense
page # 4
5’-CACATCTGCAAGTACGTTCGTTTA-3’,
5’-CAGAGCGGAGAAAGCATTTGTT-3’;
5’-AATGCGGAAGGTCATGGC-3’,
5’-CAGCTTTCCTGTCTCAGTAGCAAA-3’;
5'-AAGATGGGTCACCAGCAGCTCTACG-3',
5'-AGACGCGGCAAGAGCGAGAA-3'.
Assessment of cytokine protein expression
Tissue sampling and preparation. For joint fluid sampling, at necropsy (48 hours after arthritis
induction), the patellar ligament was cut and the articular cavity incised perpendicularly to the
patella. The synovial fluid was then collected by impregnation of small pieces of filter paper
(Schleicher & Schuell, GmbH, Germany) (4 mm2). These paper pieces were left in 150 µL of
sterile saline at 4 °C for 12 hours, and then frozen at -80 °C until processing [7]. For in vitro
studies, supernatants were collected after 18 hours of stimulation by IL-1β (10 ng/mL, R&D
Systems, Lille, France).
ELISA test and nitrite assay. The contents of the samples (cell culture supernatants and synovial
fluid) were analyzed by both NO and TNF-α dosage. NO production was determined
spectrophotometrically by measuring the accumulation of nitrites in culture supernatants by the
Griess reaction [8].
The TNF-α concentrations were determined using a sandwich ELISA (R&D System) as
previously described [9].
Multiplex immuno-assay. Levels of 24 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL10, IL-12p(70), IL-13, IL-17, IL-18, Leptin, GRO/KC, TNFα, IFNγ, GMCSF, RANTES, MCP-1,
MIP-1α, G-CSF, IP-10, Eotaxin, vEGF) were determined in synovial fluid (sensitized and
contralateral) at 7 and 24 hours and at Day 7 with the LINCOplex KIT (LINCO Research, USA).
Millipore multiscreen 96-well filter plates (Bedford, USA) for multiplex cytokine kits were used.
Assays were run in triplicate according to the manufacturers’ protocol [10]. Data were collected
using the Luminex-100 software Version 1.7 (Luminex, Austin, USA) and analysis was
performed with the MasterPlex QT 1.0 system (MiraiBio, Alameda, CA). A five-parameter
regression formula was used to calculate the sample concentrations from the standard curves.
Paquet et al.
TNF gene targeting via TFO and arthritis
page # 5
Statistical analysis
All results are expressed as mean ± SEM. All analyses and editing of figures were performed
using GraphPad Prism (release 4, GraphPad Software, Inc. San Diego, USA). Student’s t-test was
used to compare a batch to its own control and ANOVA with post-hoc Bonferroni when required
(i.e. when groups ≥ 3). The Mann-Whitney-Wilcoxon test was used for non-parametric data.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Grossin L, Cournil-Henrionnet C, Pinzano A, Gaborit N, Dumas D, Etienne S, et al. (2006). Gene
transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental
osteoarthritis. Faseb J 20: 65-75.
McDougall JJ, Watkins L, Li Z (2006). Vasoactive intestinal peptide (VIP) is a modulator of joint
pain in a rat model of osteoarthritis. Pain 123: 98-105.
Guingamp C, Gegout-Pottie P, Philippe L, Terlain B, Netter P, Gillet P (1997). Mono-iodoacetateinduced experimental osteoarthritis: a dose-response study of loss of mobility, morphology, and
biochemistry. Arthritis and rheumatism 40: 1670-1679.
van der Sluijs JA, Geesink RG, van der Linden AJ, Bulstra SK, Kuyer R, Drukker J (1992). The
reliability of the Mankin score for osteoarthritis. J Orthop Res 10: 58-61.
Rooney M, Condell D, Quinlan W, Daly L, Whelan A, Feighery C, et al. (1988). Analysis of the
histologic variation of synovitis in rheumatoid arthritis. Arthritis and rheumatism 31: 956-963.
Kirchmeyer M, Deffaud J, Sebillaud S, Moulin D, Koufany M, Netter P, et al. (2008). Evidence for
species differences in the regulation of MMPs by all-trans retinoic acid in cytokine-stimulated
chondrocytes. Biorheology 45: 415-432.
Presle N, Cipolletta C, Jouzeau JY, Abid A, Netter P, Terlain B (1999). Cartilage protection by nitric
oxide synthase inhibitors after intraarticular injection of interleukin-1beta in rats. Arthritis and
rheumatism 42: 2094-2102.
Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982). Analysis of
nitrate, nitrite, and [15N]nitrate in biological fluids. Analytical biochemistry 126: 131-138.
Szekanecz Z, Halloran MM, Volin MV, Woods JM, Strieter RM, Kenneth Haines G, 3rd, et al.
(2000). Temporal expression of inflammatory cytokines and chemokines in rat adjuvant-induced
arthritis. Arthritis and rheumatism 43: 1266-1277.
Khan IH, Krishnan VV, Ziman M, Janatpour K, Wun T, Luciw PA, et al. (2009). A comparison of
multiplex suspension array large-panel kits for profiling cytokines and chemokines in rheumatoid
arthritis patients. Cytometry 76: 159-168.