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Human Immunology 71 (2010) 342–350

Contents lists available at ScienceDirect

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uman leukocyte antigen-G molecules are constitutively expressed by synovialbroblasts and upmodulated in osteoarthritislessia Ongaro a, Marina Stignani b, Agnese Pellati a, Loredana Melchiorri b, Leo Massari c,aetano Caruso c, Monica De Mattei a, Angelo Caruso a, Olavio R. Baricordi b, Roberta Rizzo b,*

Section of Histology, Department of Morphology and Embryology, University of Ferrara, Ferrara, ItalySection of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, ItalyDepartment of Biomedical Sciences and Advanced Therapies, Orthopaedic Clinic, University of Ferrara, Ferrara, Italy

R T I C L E I N F O

rticle history:eceived 30 July 2009ccepted 15 January 2010vailable online 6 February 2010

eywords:LA-Gsteoarthritisynovial fibroblastsL-10nflammation

A B S T R A C T

Human leukocyte antigen (HLA)-G molecules are nonclassical HLA class I antigens expressed as membranebound and soluble isoforms (sHLA-G) with a restricted tissue distribution and anti-inflammatory functions.Because inflammation is involved in the pathogenesis of osteoarthritis (OA),wehave analyzed the expressionand production of HLA-G molecules in in vitro cultured synovial fibroblasts (SFs) from OA patients andcontrol subjects. We have analyzed the levels of sHLA-G1 and HLA-G5 isoforms by immunoenzymaticassay (enzyme-linked immunosorbent assay) in the SF culture supernatants from six OA patients and sixcontrol subjects in 70-day in vitro cultures and after the addition of lipopolysaccharide or recombinantinterleukin (IL)-10 (rIL-10). We have confirmed HLA-G modulation by cytofluorimetry and immunofluores-cence. The results have demonstrated the spontaneous production of sHLA-G1 molecules by both OA andcontrol SFs. The expressionwas confirmedby cytofluorimetry and immunofluorescence. OASFs produce bothsHLA-G1 and HLA-G5 molecules during the first 23 days of culture and higher levels of sHLA-G1 during thefirst 40 days of in vitro culture and after lipopolysaccharide or rIL-10 activation compared with control SFs.The production of HLA-G1 molecules, constitutively expressed by control and OA SFs, is significantly in-creased in OA, suggesting a possible mechanism to counteract the inflammation of the synovial joints.

� 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights

reserved.

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. Introduction

Osteoarthritis (OA) is a complex degenerative disease that af-ects articular cartilage components and causes damage to thentire joint structure including synovium. OA is one of the mostrevalent causes of disability in the aging population of developingountries [1]. Although the etiology of OA is not completely clari-ed, the disease development and progression were initially re-ated to mechanical and endogenous factors and more recentlyssociatedwith synovial inflammation. Acute synovitismay be onef the first changes to occur, as synovial tissue from OA patientsas shown to overexpress proinflammatory mediators such ashe interleukin-(IL)-1� cytokine and tumor necrosis factor-� [2].hese cytokines suppress the synthesis of extracellularmatrixmac-omolecules [3,4] and stimulatematrix-destructive proteases [5,6],ausing an accelerated catabolism in articular joint tissue. Thectivation of cartilage degradation in OA depends on a complexnterplay between pro- and anti-inflammatory factors and synovialnflammation contributes to the pathogenesis of OA through the

A. Ongaro and M. Stignani contributed equally.Corresponding author.

cE-mail address: rbr@unife.it (R. Rizzo).

198-8859/10/$32.00 - see front matter � 2010 American Society for Histocompatibilityoi:10.1016/j.humimm.2010.01.015

ormation of various catabolic and proinflammatory mediators,hich alter the balance of cartilage matrix degradation and repair.he anti-inflammatory molecules as IL-4, IL-10, IL-13, and IL-1�

eceptor antagonist (IL-1Ra) seem to control inflammatory damagen OA [7] and their therapeutic use is consequently one of the mostffective OA therapies.Human leukocyte antigen (HLA)-G antigens are nonclassical

LA class I molecules, which differ from classical determinants forheir limited polymorphism (44 alleles and 14 protein variants), aestricted tissue expression and the presence of mRNA splicingechanisms with the production of membrane bound (HLA-G1-4) and soluble (sHLA-G) isoforms (HLA-G5-G7). The soluble iso-orms retain the intron 4, which includes a stop codon and leads tohe termination of mRNA translation before the transmembraneomain. The HLA-G1 and HLA-G5 structures are characterized byhree heavy-chain globular domains noncovalently associatedwith2-microglobulin, in contrast to the other isoforms (HLA-G2, HLA-3, HLA-G4, HLA-G6, and HLA-G7), which lack one or more globu-ar domain and should not associate with b2-microglobulin. Theroteolytic cleavage of surface HLA-G1 isoform generates a solubleLA-G1 form (sHLAG1) [8].The HLA-G modulation was first identified in cytotrophoblast

ells and related to the induction andmaintenance of a tolerogenic

and Immunogenetics. Published by Elsevier Inc. All rights reserved.

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A. Ongaro et al. / Human Immunology 71 (2010) 342–350 343

icroenvironment at the fetomaternal interface [9]. HLA-G antigenas detected in physiologic conditions in the adult thymicmedulla,ornea, pancreatic islet, erythroid, and endothelial precursor [10]nd in plasma samples from control subjects [11]. Interferons,ormones, and IL-10 cytokine induce HLA-G modulation and theroduction of sHLA-G antigens by CD14� peripheral blood mono-ytes [8]. Several researches have evidenced the presence of HLA-Golecules also in autoimmune diseases, tumors, virally infectedells, and transplanted organs [12]. HLA-G molecules, mainlyLA-G1 and HLA-G5, can display inhibitory functions against natu-al killer cells, T lymphocytes, and antigen-presenting cell-bindingg-like transcript (ILT)-2 (LILRB1/CD85J), ILT-4 (LILRB2/CD85d), andIR2DL4 (CD158d) inhibitory receptors [13]. HLA-G1 protein canlso be expressed on the cell surface in a unique pattern ofisulfide-linked oligomers that seems to increase the avidity ofLT receptor binding [14].

HLA-G molecules, in both membrane-bound (HLA-G1) and sol-ble (sHLA-G1/HLA-G5) isoforms, are able to inhibit the cytotoxicctivity of natural killer cells and to induce T CD8(�) cell apoptosis15]. HLA-G molecules are able to inhibit CD4(�) T-cell alloprolif-rative response [16,17], to induce their differentiation into sup-ressive cells [18]. HLA-G molecules disrupt the maturation ofendritic cells and inhibit dendritic cell-triggered allogeneic T-cellroliferation [19]. Overall these characteristics propose a role forLA-G molecules in the regulation of innate and adaptive immuneesponses.

The immune cells are implicated in the synovium inflammations documented by a high CD4(�)/CD8(�) T-cell ratio in the blood ofatients comparedwith controls reflecting a possible disease-specificbnormality in T-cell response and activated/effector status [20].he presence ofmature dendritic cells in inflamed synoviummightelp to perpetuate the inflammatory condition inducing autoreac-ive T cells [21]. The presence of immunomodulatory mechanisms,s HLA-G production, could be an attempt to regulate and counter-ct synovium inflammation.To evaluate whether HLA-G1 and HLA-G5 antigens could be

roduced by synovial fibroblasts (SFs) as a possible mechanism toegulate OA inflammation, we have analyzed sHLA-G1/sHLA-G5evels in the supernatants from 70-day in vitro SF cultures fromontrol subjects and OA patients and we have characterized the SFLA-G1/HLA-G5 expression with or without an inflammatorytimulus.

. Materials and methods

.1. Patients

OAdiagnosiswas based on clinical and radiographic evaluationsccording to standard criteria [22]. We have analyzed six OA pa-ients, (two males and four females) and six control subjects with-ut inflammatory pathologies (threemales and three females). Theean age at the time of surgery was 71 � 10 years (range, 59–82ears) for OA patients and 79 � 5 years (range, 74–84 years) forontrols.SFs were isolated from fresh synovial tissue biopsy obtained

uring hip arthroprosthesis for OA patients and during hip jointeplacement surgery after femoral neck fractures for control sub-ects. Informed consent was obtained from each patient.

.2. Fibroblastlike synoviocyte cell cultures

Synovial tissue was processed within 2 hours after harvestingrom patients. After discarding fat and fibrous tissues, the syno-ium was mechanically dispersed, cut into small pieces and di-ested for 1–2 hours at 37 �C in Dulbecco’s modified Eagle mediumontaining a 1.5 mg/ml of collagenase type I-A and 1 mg/ml ofyaluronidase (Sigma-Aldrich, Milan, Italy). The cells were centri-

uged and washed after digestion [23,24]. Human fibroblastlike 6

ynoviocytes were maintained in culture in Dulbecco’s modifiedagle medium, 10% fetal calf serum, penicillin (100 U/ml), strepto-ycin (100 �g/ml), L-glutamine (2 mM) [24]. Culture medium wasollected when the cell reached sub-confluence (6400 cell/cm2).he experiments for SF characterization were performed with pri-ary cultures.

.3. mRNA preparation

Total cellular RNA was prepared from each SF culture withRIzol reagent (Life Technologies) as described [25]. The mRNAamples were digested with DNase. The quality and quantity ofRNA samples were assessed by a 1% agarose gel electrophoresis,

ollowed by ethidiumbromide staining. ThesemRNA samplesweremmediately used for cDNA synthesis or stored frozen at �80 �Cntil use.

.4. Human fibroblastlike synoviocyte characterization

.4.1. ImmunofluorescenceImmunofluorescence on primary cultures of human SFs with

he anti-human vimentin monoclonal antibody (MoAb) (Sigma-ldrich) and with anti-human CD14 (Sigma-Aldrich) was used tovaluate the expression of vimentin fibroblast marker and thebsence of contaminating macrophages, respectively. Peripherallood mononuclear cells, isolated by Ficoll centrifugation wereested for CD14 expression as positive control. One-week-old SFultures were fixed with cold methanol, washed with phosphate-uffered saline (PBS) and incubated with anti-human vimentinoAb (at 1:200 dilution) or anti-human CD14 MoAb (at 1:100ilution) for 1 hour at 37 �C. Washed slides were incubated with aecondary fluorescein isothiocyanate (FITC)�conjugated goat anti-ouse antibody (Sigma-Aldrich) for 1 hour at 37 �C in the dark.uclei were stained with the selective DNA dye, 4=,6-diamidino-2-henylindole 0.1 mg/ml in PBS-ethylene glycol tetraacetic acidSigma-Aldrich) for 10 minutes. Fluorescence was visualized usinghe Nikon Eclipse TE 2000-E microscope (Nikon Instruments Spa,esto Fiorentino, Italy) equipped with a digital camera (DXM200F; Nikon Instruments Spa).

.4.2. Reverse transcription–polymerase chain reactionsnd analysesTo confirm the absence of macrophage and endothelial cell

ontamination, the SF primary cultures were analyzed for CD14 andon-Willebrand factor (vWF) expression by reverse transcription–olymerase chain reaction (RT-PCR). Briefly, 2 �l cDNA were am-lified by specific oligonucleotide primers for CD14 (for- 5=-CTGAA GCC GGC G-3=; rev- 5=-AGC TGA GCA GGA ACC TGT GC-3=) andor vWF (for- 5=-TGGCCAGAC CTTGCTGAAGA-3=; rev- 5=-CCA TTAGG AGA ATC ACC TCC A-3=).PCR reactions were performed as previously described [24].

ycling parameters were as follows: 1 minute at 94 �C, 1 minute athe specific annealing temperature (62 �C for CD14 and 55 �C forWF), and 1 minute at 72 �C. PCR product sizes were 405 bp forD14 and 252 bp for vWF. mRNA from human macrophages andndothelial cells were used as a positive control for CD14 and vWFxpression, respectively.To analyze the presence of HLA-G mRNA in control and OA

Fs, 2 �g mRNA were reverse transcribed for each sample usinguperScript First-Strand Synthesis System (Invitrogen, San Giulianoilanese, Italy) according to manufacturer’s instructions. b-Actin

for- 5=-GCTGCTATCACTTAGACCTCA-3=; rev- 5=-CTTGTCACAGTG-AGCTCAC-3=) was used as control for the mRNA content; HLA-GRNA was evaluated by a nested RT-PCR (first round for- 5=-AACCT CTT CCT GCT GCT CT-3=; rev- 5=-CTC CTT TTC AAT CTG AGC TCTCT-3= with the cycling parameters: 40 cycles of 30 seconds at4 �C, 30 seconds at 55 �C, and 2minutes at 68 �C, final extension at

8 �C for 5 minutes; second round: for- 5=-GAG CGA GGC CAG TTC

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A. Ongaro et al. / Human Immunology 71 (2010) 342–350344

ig. 1. Vimentin andCD14 expression in human synovial fibroblast (SF) cultures by immunofluorescence. Phase contrast (A). Vimentin (B) andCD14 expression (C). Peripherallood mononuclear cells were used as positive control for CD14 (small square in Panel C). Nuclei have been counterstained in blue DAPI. Original magnification, �200. CD14xpression in human SF cultures by cytofluorimetry. (D) The SFs have been stained with anti-CD14 moAb as reported in the Material and methods section. CD14 and vWF

RNA expression by RT-PCR. (E) CD14 and vWF mRNA expression in macrophages (MC), endothelial cells (EC), and SFs. One microgram of total RNA has been loaded andtained with ethidium bromide to confirm equal RNA quantity. M � DNA ladder marker (Biolabs, Ipswich, MA).

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A. Ongaro et al. / Human Immunology 71 (2010) 342–350 345

ig. 2. Membrane andmRNAHLA-G expression by human synovial fibroblasts (SF) cultures. Immunofluorescence (A, B), flow cytofluorimetric (C, D), and RT-PCR (E) analysis

f control (CT) and OA SFs. The SFs have been stained with anti-HLA-G MoAb (MEM-G9, Exbio, Praha, CZ). Original magnification, �200 (A, B). The mean fluorescence of theeaks is reported (C, D).

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A. Ongaro et al. / Human Immunology 71 (2010) 342–350346

CA-3=; rev- 5=-AGGGAAGAC TGC TTC CAT CTC-3=, with the cyclingonditions: one cycle of 3 minutes at 94 �C and then 40 cycles of 30econds at 94 �C, 30 seconds at 58 �C, and 1 minute at 68 �C)btaining an amplified fragment of length 576 bp [26]. Jeg-3 cellRNA was used as positive HLA-G control [27].The CD14, vWF, b-actin, and HLA-G amplification products were

nalyzed by ethidium bromide–stained agarose gel electrophoresis.

.4.3. Cytofluorimetric analysis of SFsSF primary cultures were characterized for the presence of

D14-positive cells by a cytofluorimetric approachwith FACSCountow cytometer (Becton Dickinson, San Jose, CA) using standardettings and data were analyzed with CellQuest software (Bectonickinson). The anti-CD14� FITC-conjugated MoAb (Cymbus Bio-echnology, Hants, United Kingdom) was used. Anti-isotype con-rols (Exbio, Praha, Czech Republic) were used.

.5. HLA-G production

.5.1. SF culturesSFs were cultured in triplicate and the culture supernatants

ollected at days 7, 11, 16, 23, 28, 34, 41, 46, 50, 56, 60, and 70;tored at �20 �C; and then analyzed for presence of sHLA-G. Toompare the results, the SF cultures were maintained sub-confluent,ith samecellularity (6400cells/cm2) at each timepoint. In a paralleleries of SF cultures, cell viability was evaluated by Trypan bluexclusion test (Sigma-Aldrich, Irvine, United Kingdom). Briefly,ells were trypsinized from the monolayers and recovered by cen-rifugation. Cells were loadedwith Trypan blue dye and counted onhemocytometer slide under a light microscope (Nikon invertedicroscope Eclipse TE2000, Nikon Corp, Tokyo, Japan) at �100agnification.To evaluate the effect of an inflammatory stimulus, 6400 SFs/

m2 were cultured in triplicate with or without 1 �g/ml lipopoly-accharide (LPS) (Sigma-Aldrich). After 24 hours of incubation theulture supernatants were collected, stored at �20 �C, and then ana-yzed forpresenceof sHLA-G.ThedoseandtimeofLPS treatmentwerereviously determined by time-course dose-dependence experi-ents (data not shown).To evaluate the role of IL-10 in HLA-G production, 6400 SFs/cm2

ere cultured in triplicate with or without 10 ng/ml recombinantL-10 (rIL-10) (PeproTech, Rocky Hill, NJ). After 24 hours of incuba-ion the culture supernatants were collected, stored at �20 �C, andhen analyzed for sHLA-Gpresence. The dose and time of rIL-10 treat-ent were previously determined by time-course dose-dependencexperiments (data not shown).

.5.2. Membrane HLA-G antigens in SFsSFs were analyzed by a cytofluorimetric approach (with FACSCount

ow cytometer; Becton Dickinson) using standard settings andellQuest software (Becton Dickinson) for data analysis. Cells via-ility was assessed by propidium iodide staining. The membraneound HLA-G antigens were detected by anti-HLA-G FITC MoAbMEM-G/9; Exbio). Anti-isotype controls (Exbio) were used.

In addition, SFs were analyzed for the expression of HLA-Golecules by immunofluorescence assay. Briefly, SFs were washedith PBS, fixed with cold methanol-acetone 1:1 for 5 minutes,insed with PBS, and treated with 1% bovine serum albumin in PBSor 30 minutes at room temperature. Cells were incubated withhe anti-HLA-G MoAb (MEM-G9; Exbio) at 1:100 dilution for 1our at 37 �C. Washed slides were incubated with a secondaryITC-conjugated goat anti-mouse antibody (Sigma-Aldrich) for 1our at 37 �C in the dark. Nuclei were stained with the DNA dye,=,6-diamidino-2-phenylindole 0.1 mg/ml in PBS-ethylene glycoletraacetic acid for 10 minutes. Fluorescence was visualized usinghe Nikon Eclipse TE 2000-E microscope (Nikon Instruments Spa)

quipped with a digital camera (DXM 1200 F).

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.5.3. sHLA-G detection by enzyme-linked immunosorbent assaysHLA-G1 and HLA-G5 antigen concentrations were investigated

n the SF culture supernatants by enzyme immunosorbent assay, aseported in the EssenWorkshop on sHLA-G quantification [28].Wesed MoAb MEM-G9 at a concentration of 20 �g/ml as capturentibody, which recognizes HLA-G molecule, in �2-microglobulin-ssociated form (sHLA-G1/HLA-G5). An anti-�2 microglobulinoAb-HRP conjugate was used as detecting antibody.To determine the presence of HLA-G5 isoform, a further enzyme-

inked immunosorbent assay (ELISA) test was performed with 20g/ml of 5A6G7 MoAb as capture antibody, which recognizes thentrone 4- encoded epitope retained in HLA-G5 isoform, and 10g/ml of W6-32 MoAb as detecting antibody, which recognizes

2-microglobulin-associated HLA class I heavy chain.After ELISAmeasurements of sHLA-G1/HLA-G5 andHLA-G5, the

mount of sHLA-G1 was expressed by the difference betweenHLA-G1/HLA-G5 and HLA-G5 concentrations.

HeLa cells transfected with HLA-G5 (kindly provided by Prof. E.eiss, Institut fur Anthropologie und Genetik, LMU, Munchen,ermany)were cultured in CD hybridomaAGtmedium addedwith

ig. 3. sHLA-G secretion by human synovial fibroblasts (SFs). SF culture superna-ants have been analyzed for the presence of sHLA-G molecules during a 70 dayulture. Panel A reports the results obtained with anti-HLA-G MoAb (MEM-G9,xbio, Praha, CZ) in OA (�) and control (e) SF cultures. Panels B and C report theomparison between sHLA-G1 and HLA-G5 isoforms in OA and control SF culture

upernatants, respectively. ELISA technique with anti-HLA-G MoAb (5A6G7, Exbio,raha, CZ) has enabled the detection of HLA-G5 isoform.

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A. Ongaro et al. / Human Immunology 71 (2010) 342–350 347

% FCS and antibiotics. Culture supernatants were collected at cellonfluence and concentrated by lyophilization. Following deple-ion of albumin by Albumin depletion kit (Enchant Life Science kit;all Corporation, Ann Arbor, MI), the purification of the sHLA-Groteins was carried out as previously reported [29]. The sHLA-Golecules obtained were used as standard at different dilutions.The intra- and inter-assay coefficients of variation are 1.4% and

.0%, respectively. The limit of sensitivity is 1.0 ng/ml.

.5.4. IL-10 detection by ELISA assayIL-10 concentrationswere determined in triplicate using the com-

ercially availableHuman IL-10BioSource ImmunoassayKit (HumanL-10US; BioSource, Camarillo, CA)with adetection limit of 0.2 pg/ml.

.6. Statistical analysis

Statistical analysis was conducted using the Stat View softwareackage (SAS Institute, Cary, NC). The data were analyzed by theann–Whitney U test. The correlation between variables was as-essed by the Spearman correlation test. Statistical significancewasssumed for p � 0.05 (two-tailed).

. Results

.1. SF characterization

Cell morphology, evaluated by light microscopy, showed a typ-cal fibroblastlike spindle shaped form (Fig. 1A). Primary human SFultures have shown vimentin expression, a specific cellulararker for mesenchymal cells and fibroblasts (Fig. 1B). No stainas observed with anti-CD14 MoAb by immunofluorescence (Fig.C) and cytofluorimetric assay (Fig. 1D), indicating the absence ofacrophage contamination. RT-PCR for CD14 and vWF has ex-luded macrophage and endothelial cell contamination in SF cul-ures (small square in Panel C). As CD14(�)monocytes are themainroducer of HLA-G molecules it is extremely important to haveure SF cultures to ensure that the revealed HLA-G moleculeserive from SF cells.

.2. Membrane HLA-G modulation in SF culture

The presence of membrane HLA-G molecules was observed bymmunofluorescence and cytofluorimetry (Fig. 2). MembraneLA-G molecules are present in both control (A, B) and OA (C, D);Fswith a higher expression ofHLA-G (meanfluorescence intensityFI]: 51.15) in OA in comparison with control subjects (mean FI:

ig. 4. Membrane HLA-G expression by human synovial fibroblasts (SFs). OA SFs have beeays of culture. The SFs have been stained with anti-HLA-G MoAb (MEM-G9, Exbio, Praha

6.11). HLA-G specific mRNA was detected in both control and OAFs (E).

.3. Soluble HLA-G in SF cultures

SFs were cultured for 70 days and the culture supernatantsnalyzed for presence of sHLA-G at different time points. In SFultures, the 95 � 3% of cells were viable at all time points, asvaluated by Trypan blue assay (data not shown). In Fig. 3A theHLA-G levels in the SF culture supernatants from OA and controlubjects are reported. The OA SFs present significant higher levelsf sHLA-G during the first 40 days of culture in comparison withontrol subjects (p � 0.024;Mann–WhitneyU test). In Fig. 3B, C theHLA-G1 and HLA-G5 levels are reported in both OA and control SFulture supernatants. The control SFs secreted only HLA-G1 iso-orm while HLA-G5 accounted the 50% of the secreted HLA-G dur-ng the first 23 days of OA SF culture; then the only secreted isoforms HLA-G1. Themembrane HLA-Gwas evaluated on OA SFs after 30ays of culture, which correspond to the highest HLA-G secretion,nd 70 days of culture, which correspond to the lowest sHLA-Gevel (Fig. 4). The membrane HLA-G expression significantly de-reased from30days (mean FI: 35.05) to 70days (mean FI: 12.74) ofulture according to the sHLA-G results.

.4. IL-10 in SF cultures

SFs were analyzed during 70 days of culture for presence ofL-10. In Fig. 5A the IL-10 levels in the SF culture supernatants fromA and control subjects are reported. The OA SFs produce detect-ble levels of IL-10 during the first 30 days of culture while controlubject SFs produced no IL-10 (p � 0.0001; Mann–Whitney U test).he levels of sHLA-G and IL-10 molecules in OA SF supernatantsresent a significant correlation (p � 0.007, r � 0.818, Spearmanorrelation test) (Fig. 5B).

.5. IL-10 treatment of SF cultures

SFs proliferation was evaluated during an 8-day culture fromhe sub-confluencepoint. The results (Fig. 6) confirmed the absencef a decrease in SF viability and the possibility to analyze HLA-Gecretion during a 24-hour culture. The ability of IL-10 to induceHLA-G secretion was evaluated adding 10 ng/ml rIL-10 to un-reated OA and control SF cultures for 24 hours. rIL-10 has inducedHLA-G production in SF cultures without any statistical differencen sHLA-G levels between OA and control SFs (Fig. 7).

n analyzed for the presence of membrane HLA-G molecules after 30 (A) and 70 (B), CZ). The mean fluorescence of the peaks is reported.

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.6. LPS treatment of SF cultures

SFs were analyzed for sHLA-G production after LPS treatment,n inflammatory stimulus able to induce sHLA-G production30]. LPS was used at 1 �g/ml dose for 24 hours. These experi-ents have enabled the analysis of SF secretion in an inflamma-

ory microenvironment.Untreated SFs have presented sHLA-G secretion with lower lev-

ls in control SF cultures (p � 0.02; Mann–Whitney U test), whilePS treatment have increased sHLA-G secretion obtaining the sameevels in both OA and control SFs (Fig. 8). These results show that

ig. 5. IL-10 secretion by human synovial fibroblasts (SFs). IL-10 has been analyzedy Human IL-10 BioSource Immunoassay Kit (Human IL-10 US; BioSource, Cama-illo, CA) (A). IL-10 and sHLA-G levels in OA SF cultures present a significant corre-ation (p � 0.007, r � 0.818, Spearman correlation test) (B).

ig. 6. Cell proliferation of synovial fibroblasts (SF) cultures. Cells proliferation has2

een evaluated during the 8 days from the sub-confluence (6400 cell/cm ) point, by

rypan blue exclusion test.LG

oth OA and control subjects SFs are able to produce sHLA-G mol-cules after an inflammatory stimulus.

. Discussion

This study has evidenced for the first time the expression androduction of membrane and soluble HLA-G molecules by SFs.

ig. 7. Soluble HLA-G expression by human synovial fibroblast (SF) cultures treatedith rIL-10. OA (�) and control (e) SF cultures have been analyzed for sHLA-Gontent without (w/o rIL-10) and with addition of rIL-10 (rIL-10). About 10 ng/mlIL-10 has been added at each SF culture. ELISA technique with anti-HLA-G MoAbMEM-G9, Exbio, Praha, CZ) detected sHLA-G. * Mann–Whitney U test.

ig. 8. Soluble HLA-G expression by human OA (�) and control (e) synovial fibro-last (SF) cultures with (LPS) and without LPS treatment (w/o LPS). About 1 �g/ml

PS has been added at each culture. ELISA technique with anti-HLA-GMoAb (MEM-9, Exbio, Praha, CZ) detected sHLA-G. * Mann–Whitney U test.

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To exclude the presence of a monocyte contamination we ana-yzed the expression of CD14� molecules by RT-PCR, immunofluo-escence, and flow cytofluorimetry. The absence of monocytes hasxcluded that HLA-G expression might be due to contamination.Weobserved thepresenceofHLA-G–specificmRNAandmembrane-

ound HLA-G molecules in both OA and control SFs with higherevels in OA patients as documented by the flow cytofluorimetryhat has evidenced higher percentages of HLA-G–positive SFs in OAamples in comparison with control subjects. The same SFs werenalyzed for the presence of sHLA-G in the culture supernatants.e observed sHLA-G in both OA and control subject SF culture

upernatants with higher levels in OA patients. To verify a possiblehange in HLA-G expression during the in vitro culture, we per-ormed a longitudinal evaluation during a 70-day culture. We ob-erved the presence of sHLA-G molecules both in control subjectnd OA patient SFs during the whole period even though with arogressive decrease. Interestingly, theOASF supernatants showedignificantly higher concentrations of sHLA-G molecules in com-arisonwith control subjects. Themain difference in sHLA-G levelsas observed during the first 40 days of culture, while in the last 20ays of culture the sHLA-Gmolecules presented similar concentra-ions in OA and control subject supernatants. The membraneLA-G expression significantly decreased from 30 to 70 days ofulture according to the sHLA-G results. These results might sup-ort the hypothesis of an in vivo inflammatory microenvironmentn OA patients that induces an increased membrane and solubleLA-Gproduction by SFs. TheOASF couldhave an activated inflam-atory status that is maintained during the first 30–40-day cul-

ure. The progressive downregulation of sHLA-G production by SFsould be due to the loss of an inflammatory microenvironment inhe in vitro culture, as the 70 day OA SFs have a similar behavior inomparison with control SFs. SFs under continuous stimulationith tumor necrosis factor-� and IL-1� are able to degrade intactartilage matrix by disturbing the homeostasis of cartilage via pro-uction of catabolic enzymes/proinflammatory cytokines and sup-ression of anabolic matrix synthesis [31]. The absence of theytokine treatment reduces the SF activity toward the cartilage,ocumenting the importance of an inflammatory microenviron-ent to induce SF activation.The evaluation of the HLA-G isoform secreted by SFs has docu-

ented the presence of HLA-G1 isoform in control SF culture su-ernatants while HLA-G5 accounted the 50% of the secreted HLA-Guring the first 23 days of OA SF culture. These results couldonfirm the effect of a specific microenvironment to sustainLA-G5 production, which is controlled by a specific mRNAplicing mechanism.Interleukin-10, a pleiotropic cytokine with anti-inflammatory

roperties, is negatively correlated with joint destruction [32]. Theresence of elevated mRNA and protein levels of this cytokine haseen previously reported in osteoarthritic chondrocytes and SFsuggesting an anti-inflammatory role for IL-10 in synovial inflam-ation [33]. Moreover, IL-10 is the main up-modulator of sHLA-Groduction by CD14(�) peripheral bloodmonocytes. No detectableevels of IL-10 were observed in control SFs while this cytokine isresent in OA SF supernatants during the first 30 days of culture. Aignificant correlation was observed between IL-10 and sHLA-Gevels in OA patients. These results suggest that IL-10 could have aegulatory effect on sHLA-G production in OA SFs sustaining theigh sHLA-G levels observed in the first 40 days of culture. The lackf IL-10 in control SF cultures confirms the absence of a chronicnflammation in these specimens. To verify this point we havenalyzed the ability of IL-10 to induce sHLA-G secretion by SFs.ecombinant IL-10 addition to SF cultures has increased sHLA-Gecretion in both OA and control subject SFs. It is known that IL-10s one of the main up-modulators of sHLA-G production and the

nflammatory microenvironment that is present in the OA joints

ould justify the secretion of IL-10 and consequently of sHLA-G as arotection mechanism.To recreate an in vitro inflammatorymicroenvironmentwehave

reated SFs with LPS, one of themain inducer of HLA-G production.e have observed a significant increase in sHLA-G levels in bothOA

nd control subject LPS-treated SF cultures. These data propose thenflammatory stimuli as inducer of SF sHLA-G production.

Even though these results are still preliminary and the numberf SF cultures should be increased, the difficulties in obtaining andulturing these specimens make this study interesting, as theyupport the hypothesis of an HLA-G molecule production via IL-10pmodulation during synovial inflammation. The presence ofLA-G molecules seems to be an effort to limit and contrast theevelopment of this condition. HLA-G immunoinhibitory functionsould be implicated in the regulation of CD4(�)/CD8(�) T-cell rationd T-cell response that are impaired during synovial inflamma-ion [20]. These new data could help elucidate the role of SFs inartilage in vivo destruction and the molecular mechanisms in-olved in this process. The real significance of theHLA-Gexpressionn osteoarthritis could be evaluated by analyzing patients with aifferent OA grades. This could explain whether the presence ofLA-Gmolecules is a positive or a negative event. It iswell acceptedhat HLA-G expression is beneficial during embryo implantationnd organ transplantation, while it is associated with deleteriousvents in cancer and viral infections. In inflammatory diseases,lthough the presence of contrasting results, the expression ofLA-G antigens seems to positively counteractNKand autoreactivecells, inducing the production of HLA-G–positive regulatory T

ells. We could speculate on a possible positive effect so as toontrol the chronic inflammation, but further studies are necessary.

cknowledgments

This work was supported in part by the Fondazione CARICENTOFerrara, Italy).

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