The Journal of Rheumatology Volume 28, no. 4 Expression of ... · (Atlanta Biologicals, Atlanta,...

The Journal of Rheumatology 2001; 28:4698

From the Clinical Immunology Rheumatology Unit, University ofRochester School of Medicine and Dentistry, Rochester, New York, USA.

Supported by grants from the National Psoriasis Foundation and theGenesee Valley Chapter of the Arthritis Foundation.

C. Ritchlin, MD, Associate Professor of Medicine; S.A. Haas-Smith, BA,Research Associate.

Address reprint requests to Dr. C. Ritchlin, Clinical ImmunologyRheumatology Unit, University of Rochester School of Medicine andDentistry, 601 Elmwood Avenue, Box 695, Rochester, NY 14642. E-mail: [email protected]

Submitted March 17, 2000 revision accepted October 26, 2000.

In rheumatoid arthritis (RA) the synovial membrane istransformed into a proliferating cell mass or pannus thaterodes surrounding tissue and bone1. Proinflammatorycytokines released by infiltrating mononuclear cells andlining synoviocytes promote and sustain inflammation byboth autocrine and paracrine pathways2. Data derived fromclinical trials and the analysis of synovial tissues support theconcept that the proinflammatory cytokines interleukin 1ß(IL-1ß) and tumor necrosis factor-α (TNF-α) are pivotalmolecules involved in the sustained synovial inflammationand tissue destruction in the rheumatoid joint3. This intensesynovial cell hyperplasia and proliferation is associated witha compensatory antiinflammatory response characterized bythe production of soluble TNF receptors, transforming

growth factor (TGF)-ß, IL-1 receptor antagonist (IL-1RA),and IL-104.

IL-10 is a 35 kDa homodimeric cytokine produced byhuman T cells, B cells, and monocytes5. It has been shownthat IL-10 blocks inflammation via several mechanisms.First, IL-10 potently suppresses the production of proin-flammatory cytokines by Th1 lymphocytes [interferon-γ(IFN-γ), IL-2] and monocytes (IL-1, IL-6, TNF-α). Second,IL-10 reduces antigen induced T cell proliferation by down-regulation of HLA-DR expression in monocytes6. Third, IL-10 can suppress inflammation through the induction of theIL-1 receptor antagonist and soluble TNF receptors(sTNFR)7,8. Finally, IL-10 can limit bone resorption byinhibiting matrix metalloproteinases and increasing theproduction of tissue inhibitor of metalloproteinases(TIMP)9.

Levels of IL-10 are elevated in the serum and synoviumof patients with RA10 and biologically significant quantitiesof functionally active IL-10 are released in rheumatoidsynovial cell suspension cultures11. Immunostaining ofrheumatoid membranes by Katsikis, et al suggests thatcellular sources of IL-10 are monocytes in the lining layerand T cells present in the mononuclear aggregates6.However, studies in our laboratory revealed a prominent

Expression of Interleukin 10 mRNA and Protein bySynovial Fibroblastoid CellsCHRISTOPHER RITCHLIN and SALLY A. HAAS-SMITH

ABSTRACT. Objective. To determine if fibroblast-like synoviocytes (FLS) express interleukin 10 (IL-10) mRNAand protein and functional IL-10 receptors.Methods. The pattern of IL-10 production was analyzed in inflammatory synovial tissues byimmunohistochemistry. Expression of IL-10 mRNA and protein was determined by Northern blotanalysis and ELISA in resting FLS and following stimulation with IL-1ß and tumor necrosis factor-α (TNF-α). IL-10 receptor expression was measured on cultured FLS by immunohistochemistry andFACS analysis after treatment with biotinylated IL-10. Responsiveness of FLS to IL-10 was deter-mined by multigene assay and inhibition of prostaglandin E2 induced morphologic changes.Bioactivity was confirmed by downregulation of interferon-γ stimulated HLA-DR expression byFACS and inhibition of TNF-α production by U937 cells.Results. Using immunohistochemistry, we detected IL-10 in the lining layer of inflamed synovialtissue and FLS. ELISA on unstimulated third passage FLS culture supernatants revealed IL-10production that varied over time and among cell lines. FLS produced IL-10 mRNA constitutively.IL-10 production was upregulated by IL-1ß and TNF-α. IL-10 bound to receptors on FLS andinduced functional changes. Endogenously released FLS IL-10 was biologically active.Conclusion. Mesenchymal lining cells in the inflamed joint produce IL-10. In addition, culturedFLS constitutively produce IL-10 mRNA and protein that is bioactive and can be upregulated by IL-1ß and TNF-α. FLS express functional IL-10 receptors. These results suggest that IL-10 released bymesenchymal cells in inflammatory arthritis can modulate synovial inflammation and joint destruc-tion by paracrine and/or autocrine mechanisms. (J Rheumatol 2001;28:698-705)

Key Indexing Terms:INTERLEUKIN 10 SYNOVIAL FIBROBLASTS RHEUMATOID ARTHRITIS PSORIATIC ARTHRITIS

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pattern of IL-10 staining evenly dispersed throughout thesynovial lining, indicating that type B synoviocytes ofmesenchymal lineage can also produce this cytokine. In thisstudy, we demonstrate that cultured fibroblast-like synovio-cytes (FLS) constitutively express IL-10 mRNA and biolog-ically active protein along with functional IL-10 receptors(IL-10R). Furthermore, release of IL-10 is upregulated byTNF-α and IL-1ß. Thus, mesenchymal cells in the rheuma-toid joint may modulate joint inflammation through therelease of IL-10.

MATERIALS AND METHODSCell cultures. Synovial fibroblastoid cell lines were established from tissuesamples obtained from patients with inflammatory synovitis, including 6with RA and 7 with psoriatic arthritis, at the time of joint replacement orarthroscopic surgery. Cell lines were grown out of explant cultures or estab-lished by enzymatic digestion of whole synovium. Dermal fibroblasts weregrown from explants obtained by punch biopsies of normal skin. Peripheralblood mononuclear cells (PBMC) were isolated on Ficoll-Paque(Pharmacia, Piscataway, NJ, USA) gradients by established methods12.Cultures were maintained in 10% fetal bovine serum (FBS) in DMEM-F12(Atlanta Biologicals, Atlanta, GA, USA) supplemented with 2 mM L-glut-amine, penicillin (1000 units/ml), gentamycin (100 µg/ml), and strepto-mycin (10 µg/ml). The media was changed twice weekly. Cultures werepassaged when confluent using trypsin-EDTA. Third passage cultures weregrown in 175 cm2 tissue culture flasks for RNA isolation and 16 mm diam-eter tissue culture wells for ELISA and flow cytometry. Cells were grownin chamber slides for immunohistochemistry and electron microscopystudies. All cultures were maintained at 37°C and 5% CO2.

ELISA for IL-10. Confluent third passage cultures were changed to mediacontaining 1% FBS for 48 h prior to cytokine stimulation. IL-1ß or TNF-αwas added to a final concentration of 1–10 ng/ml. Unstimulated controlswere incubated in parallel to stimulated cultures. Following incubation,supernatants were harvested from duplicate wells, pooled, and stored at–70°C until assayed. Assays for IL-10 were carried out by ELISA usingmatched antibody pairs (Endogen, Cambridge, MA, USA) following themanufacturer’s protocol. Maxisorb microtiter plates (Nunc, Denmark) werecoated with rat anti-human IL-10 (clone #9D7) at 1 µg/ml in phosphatebuffered saline (PBS). Biotinylated detecting antibody rat anti-human IL-10 clone #12G8 was incubated with samples at 0.5 µg/ml. Streptavidin-HRP (Zymed, San Francisco, CA, USA) diluted 1:6000 was used as theconjugate, followed by TMB One Step Substrate System (Dako,Carpinteria, CA, USA). Recombinant human IL-10 (Endogen) was seriallydiluted in culture media, as the standard. IL-10 production is expressed aspg/ml.

Immunohistochemistry and flow cytometry. Synovial tissues wereembedded in OCT (Sakura Finetek, Torrance, CA, USA) and snap frozenin liquid nitrogen. Tissues cut in 8 mm sections were fixed in 4%paraformaldehyde and stained following the methods of Andersson, et al13.Third passage cells grown in chamber slides were used for immunohisto-chemistry. These cells were fixed in paraformaldehyde and stained asabove. Monoclonal rat anti-human IL-10 clone #9D7 (Endogen) and poly-clonal rabbit anti-human IL-10 (Genzyme, Cambridge, MA, USA) wereused as primary antibody. Specificity for IL-10 was determined by incu-bating the primary antibody with excess IL-10 (10 µg/ml) prior to staining.Rabbit anti-mouse IgG was used as the negative isotype control. Mouseanti-rat or goat anti-rabbit, either FITC or peroxidase conjugated, werepurchased from Sigma (St. Louis, MO, USA) and used as secondary anti-bodies.

Biotinylated IL-10 (R&D Systems, Minneapolis, MN, USA) was usedto detect IL-10 receptors. Third passage cultures were treated with 10 mMEDTA, incubated with biotinylated IL-10, followed by FITC conjugated

streptavidin. Cells were analyzed by FACS and visualized by fluorescencemicroscopy. Biotinylated soy trypsin inhibitor was used as the negativecontrol. Receptor staining was blocked using anti-IL-10 antibody.

To determine the lineage and purity of fibroblast cell lines, thirdpassage cells were treated with 10 mM EDTA in PBS and stained with theT lymphocyte marker anti-CD2 (clone M0720) and the monocyte markersanti-CD14 (clone TUK4) and anti-CD45 (clone T29/33; Dako) andanalyzed by flow cytometry.

Electron microscopy. Cells were cultured in multi-well chamber slides asdescribed above. Once confluent, the cells were fixed in 2.5% phosphatebuffered glutaraldehyde and postfixed in 1.0% osmium tetroxide, dehydra-dated through a graded series of ethanol, and infiltrated with Spurr epoxyresin. The cells were embedded on the glass surface with inverted capsularmolds in fresh Spurr resin and “popped-off” following polymerization at70°C14. Sections were stained with uranyl acetate and lead citrate andexamined with a Hitachi 7100 electron microscope.

Northern blot analysis. RNA was isolated from confluent third passagecultures following 48 h incubation in 1% FBS media with and without 1ng/ml IL-1ß stimulation, using RNAstat reagent (Tel-Test-B, Friendswood,TX, USA). The RNA was size fractionated in 1.2% agarose gel with 2.2 Mformaldehyde and transferred to Nytran (Schleicher and Schull, Keene,NH, USA). GAPDH and IL-10 cDNA obtained from ATCC (Rockville,MD, USA) were radiolabeled by the random primers method (Pharmacia)and hybridized overnight at 42°C. Blots were washed in 0.1% SDS 0.1 ×SSC at 60°C. Hybridization was visualized by autoradiography. The IL-10signal was normalized to GAPDH using image densitometry. Signal dataare expressed as absorbence units.

IL-10 bioactivity. The effect of IL-10 on FLS mRNA production was deter-mined by multigene analysis. Third passage FLS, grown to confluence in75 cm2 culture flasks, were changed to 1% FBS media for 24 h. Cultureswere stimulated with and without IL-10 (10 ng/ml) for 18 h. The cells werelysed and RNA isolated as for Northern blotting described above.Multigene analysis was carried out as described15. Probes for IL-1ß, IL-6,collagenase, DRß, and GAPDH were obtained from the ATCC.Hybridization was visualized by autoradiography. The probe signals werenormalized to GAPDH using image densitometry. Signal data areexpressed as absorbence units.

The effects of IL-10 on prostaglandin E2 (PGE2) induced stellatemorphology16 was assessed. Confluent third passage FLS grown in 16 mmwells were cultured in 1% FBS-DMEM 24 h prior to addition of PGE2 (500ng/ml) (Sigma) with and without IL-10 (10 ng/ml) (Endogen). Stellate cellswere scored following 8 h incubation. Cells with 5 or more dendrites werecounted as positive. Cells were counted in 5 separate low power (10×)fields (about 1000 cells).

To measure the biological activity of endogenously produced IL-10,supernatants were harvested from third passage cultures. IL-10 levels weredetermined by ELISA as described above. Supernatants were then added toIFN-γ (100 U/ml) (Endogen) stimulated FLS with and without anti-IL-10antibody (10 µg/ml) (Endogen) for 48 h. The cells were harvested with 10mM EDTA in PBS and stained with anti-HLA-DRß (Becton-Dickinson,Burlingame, CA, USA), and analyzed by FACS. In separate experimentsculture supernatants were added to U937 cell cultures (ATCC) with andwithout IL-10 blocking antibody. U937 culture supernatants were harvestedafter 48 h and TNF-α was measured by ELISA (matched antibody pairs;Endogen).

RESULTSIL-10 immunohistochemistry of inflammatory synovialmembranes and cultured fibroblastoid cells. The pattern ofIL-10 expression in 7 synovial membranes (4 RA, 3 psori-atic arthritis) was determined by immunohistochemistry.Staining with anti-IL-10 antibody revealed prominentstaining throughout the lining layer in all specimens and was

Ritchlin and Haas-Smith: IL-10 in synovial fibroblasts 699





not seen in sections stained with the isotype control (Figure1). There was very little IL-10 immunoreactivity detected inthe subsynovial tissues. We were able to confirm the validityof the immunohistochemistry by specifically blocking thereaction by preincubation of the primary antibody with IL-10 (data not shown).

Cells from third passage cultures were similarly stained todetermine if FLS could produce IL-10 in vitro. Thirdpassage FLS stained strongly for IL-10 (Figure 2). PBMCstained positively for IL-10, whereas dermal fibroblasts didnot exhibit this property.

Mesenchymal origin of cultured cells. Third passage FLSwere stained with the lymphocyte and monocyte markersanti-CD2, anti-CD14, and anti-CD45 and examined byFACS to determine the purity and cell lineage. Surfaceexpression of all these markers was undetectable (data notshown). The ultrastructural characteristics of these cellswere studied by electron microscopy. A homogeneous popu-lation of cells was observed with morphologic featuresconsistent with mesenchymal cells (Figure 3). Thesefeatures included a spindle shape appearance, heteroge-neous nuclear chromatin, a well developed endoplasmicreticulum, and the presence of large numbers of lysosomesand dense granules17,18.

IL-10 protein and mRNA production by FLS. IL-10 proteinproduction in unstimulated cells was measured by ELISA atspecific time points ranging from 2 to 48 hours and at Day10. The levels of IL-10 secretion varied between the

different fibroblastoid lines and fluctuated over time in the 4lines studied (Figure 4), but IL-10 secretion was markedlydiminished by Day 10. Six FLS lines that did not constitu-tively release IL-10 were stimulated with IL-1ß (1 ng/ml)

Figure 1. IL-10 immunohistochemistry of a synovial membrane isolated from a patient with inflammatory synovitis. Synovial tissues harvested from patientswere fixed and stained with IL-10. IL-10 staining is most intense throughout the synovial lining layer (A), while no tissue staining was detected with theisotype control (B). (Magnification 100×)

Figure 2. IL-10 immunohistochemistry of third passage FLS. Third passageFLS were immunostained for IL-10. IL-10 is present in the cytoplasm.(Magnification 100×)

A B




and secretion of IL-10 was measured at 18 h (Figure 5). IL-10 levels increased (7–37 pg/ml) in all lines after treatmentwith IL-1ß. Similar results were observed following expo-sure to TNF-α (data not shown).

IL-10 mRNA levels in third passage FLS were deter-mined by Northern blot analysis using third passage FLS celllines after 48 h in culture (Figure 6A). As expected, mRNAlevels varied in the 3 lines assayed. PBMC and dermalfibroblasts were used as positive and negative controls,respectively. Following treatment of FLS with IL-1ß, wecould detect an upregulation of IL-10 mRNA (Figure 6B).

FLS express functional IL-10 receptors. To determine if FLSexpress IL-10 receptors, we stained third passage rheuma-toid FLS with FITC labeled IL-10 (Figure 7). The labeledIL-10 specifically bound to the cell surface, while stainingwith an irrelevant biotinylated protein was undetectable. IL-10 binding was blocked by pretreatment with IL-10 anti-body. A similar staining pattern was noted with PBMC, butnot dermal fibroblasts. The presence of IL-10 receptors wasrevealed by flow cytometry (Figure 7A) and fluorescencemicroscopy (Figures 7B, 7C).

The effect of IL-10 on FLS gene expression was deter-mined using a multigene assay. Levels of IL-1ß, IL-6, colla-genase, and DRß mRNA were measured before and afterstimulation with IL-10 in third passage FLS (Figure 8). IL-10 reduced the expression of IL-1ß (1.92 to 0.34 absorbanceunits, au), IL-6 (86.5 to 0.11 au), collagenase (0.77 to 0.02au), and DRß (6.12 to 0.05 au). In addition, IL-10suppressed PGE2 induced FLS stellate cell formation in 4FLS lines by 25–40% (data not shown). These experimentsindicate that IL-10 acting through its receptor suppressesproinflammatory cytokine and HLA-DR mRNA expressionand prostanoid induced phenotypic alterations in FLS.

Bioactivity of IL-10 released by FLS. The ability of FLS IL-10 to downregulate HLA-DR expression on IFN-γ stimu-lated fibroblasts was examined by FACS analysis. FLSculture supernatants harvested at 24 h were added to thirdpassage IFN-γ stimulated FLS, and HLA-DR expressionwas analyzed by FACS analysis. Supernatant with elevatedIL-10 (80 pg/ml) inhibited expression of DRß by 16%. The

Figure 3. Electron micrograph of FLS showing mesenchymal phenotype.Clearly visible are classic spindle shape, heterogeneous nuclear chromatin,well developed endoplasmic reticulum, numerous lysosomes, and densegranules. (Magnification 1875×)

Figure 4. IL-10 production by unstimulated FLS and peripheral blood mononuclear cells (PB). IL-10 proteinlevels in culture supernatants harvested at 2, 8, 24, and 48 hours were measured by ELISA. Data are means ± SDof 4 SF lines and fresh PB from 2 donors expressed as pg/ml.






Figure 5. IL-1 stimulated IL-10 production in FLS lines. Six FLS cell lines, which produce little or no IL-10, inunstimulated cultures after IL-1ß stimulation (1 ng/ml) for 18 hours. IL–10 levels were determined by ELISA;data are expressed as pg/ml.

Figure 6. A. Northern blot analysis of IL-10 mRNAlevels in unstimulated FLS from 3 lines isolated frominflammatory synovitis, peripheral blood mononu-clear cells (PB), and dermal fibroblasts (DF). FLSand PB but not DF express IL-10. B. Upregulation ofIL-10 mRNA levels in FLS after 48 h IL-1ß stimula-tion (1 ng/ml) from 2 FLS lines. Data are expressedas absorbance units for the IL-10 signal normalizedto GAPDH.

A

B




addition of anti-IL-10 antibodies blocked supernatant inhi-bition of HLA-DR expression. Supernatant with nodetectable IL-10 did not suppress DRß (data not shown). Inseparate experiments, FLS culture supernatant added toU937 cell cultures decreased baseline TNF-α production by50%, while supernatant from a non-IL-10 producing FLSline had no effect (data not shown). Thus, endogenouslyreleased FLS IL-10 is bioactive.

DISCUSSIONIL-10 is a pivotal immunoregulatory molecule that exertsantiinflammatory actions through the inhibition of mono-

kine and lymphokine synthesis and suppression of mono-cyte HLA-DR expression. Significant levels of IL-10 arepresent in rheumatoid, psoriatic, and osteoarthritic but not innormal synovial tissues6,19. Studies have shown IL-10production by infiltrating mononuclear cells and type Asynoviocytes in the synovial lining layer6. In this study, weshow that IL-10 is present throughout the synovial lininglayer in vivo and also in serially cultured FLS isolated frominflamed joints. Fibroblast-like synoviocytes constitutivelyexpressed IL-10 mRNA and released bioactive IL-10. IL-10protein production was variable, but strikingly elevated insome cell lines. Both IL-1ß and TNF-α stimulated IL-10

Figure 7. A. FACS analysis of IL-10 receptors in third passage unstimu-lated FLS shown by FITC labeled IL-10 staining (black) compared to anirrelevant control. B and C. Fluorescence imaging of IL-10 receptors byFITC labeled IL-10. B: magnification 100×; C: magnification 450×.

A

B C






production in lines that did not spontaneously release thiscytokine. Further, IL-10 bound to IL-10 receptors on the cellsurface of FLS, inhibiting proinflammatory cytokine geneexpression and morphologic alterations induced by PGE2.

Recent data suggest that FLS in the rheumatoid jointpromote invasive synovitis by releasing proinflammatorycytokines that drive the proliferation and activation ofsynoviocytes and stimulate degradative enzyme release,resulting in erosion of surrounding tissue and bone20. Invitro, FLS cultured from rheumatoid synovium expressvascular cellular adhesion molecule-1 at high levels, consti-tutively release proinflammatory cytokines in longtermculture, and induce formation of pannus-like tissue in carti-lage explants co-implanted into SCID mice15,21-23. FLSisolated from osteoarthritis synovium do not exhibit theseproperties. Conversely, these cells can also dampensynovitis and block joint erosion through the release ofsoluble TNF receptor molecules, TGF-ß, IL-1RA, andTIMP22,24-27. This antiinflammatory phenotype must now beexpanded to include secretion of IL-10.

The levels of IL-10 released varied considerably betweenthe FLS lines and the PBMC isolated from different indi-vidual patients. Potential reasons for this variability includediffering levels of constitutive cytokine release into theculture media, alterations in level of cell surface cytokinereceptor expression (IL-1, IL-10), or constitutional differ-ences in IL-10 production resulting from polymorphisms inthe IL-10 promoter28,29. Levels of in vitro IL-10 release wererelatively high (up to 200 pg/ml) in many FLS lines isolatedfrom patients with inflammatory synovitis. In aggregate,FLS may release large quantities of IL-10, since themesenchymal cell population is greatly expanded in theinflamed synovial membrane. Moreover, activated fibro-blasts migrate to the cartilage-pannus junction, and IL-10

released by these cells may exert local downregulatoryeffects on synovial inflammation and bone resorption.

The bioactivity of endogenously released FLS IL-10 wasrevealed by antagonism of IFN-γ induced HLA-DR expres-sion in FLS and TNF-α release in U937 cells. These exper-iments may underestimate IL-10 bioactivity, sincesupernatants were isolated from unstimulated FLS, yieldingIL-10 levels that were relatively low (60–80 pg/ml). It iscertainly plausible that in the inflamed synovial membrane,mesenchymal cells release elevated quantities of IL-10 inresponse to persistent proinflammatory cytokine stimula-tion.

The mechanism of IL-10 activation in FLS or othermesenchymal cells is not known. In lipopolysaccharidestimulated monocytes, induction of IL-10 proceeds via botha TNF dependent and a TNF independent pathway. TNF-αstimulation directly promotes IL-10 release in monocytes,but the constitutive secretion of IL-10 in cultures free ofmononuclear cells suggests the presence of TNF indepen-dent mechanisms. The TNF independent pathway is medi-ated through cyclic AMP30. The response to IL-1ß supportsa role for this cytokine in induction of mesenchymal IL-10,possibly through cAMP activation. Future studies arerequired to determine the molecular mechanisms thatunderlie IL-10 production in FLS.

Our data indicate that synovial fibroblastoid cells canproduce the antiinflammatory cytokine IL-10 in vivo and invitro. Further, these cells are capable of responding to IL-10by binding to IL-10 receptors present on the cell surface ofFLS. This suggests that IL-10 can stimulate mesenchymalcells via both paracrine and autocrine interactions. Thesefindings may have implications for the development oftreatment strategies in inflammatory arthritis, although animportant question that remains to be answered is whetherthe IL-10 released into inflamed joints is either inactive orpresent in insufficient quantities to block the proinflamma-tory cascade. Presumably, IL-10 is part of an inadequatecompensatory antiinflammatory response in the synovialmembrane, since proinflammatory cytokine interactionspredominate in the diseased joint. In particular, it is conceiv-able that therapies directed toward increasing IL-10 by thesynovial fibroblastoid cell may attenuate inflammation andlimit destruction in inflammatory synovitis.

ACKNOWLEDGMENTThe authors acknowledge Karen Jensen, Aimee Pugh, and Jeff Goater fortheir expert technical assistance with electron microscopy and flow cytom-etry; the Orthopedic Surgery Departments at Rochester General and StrongMemorial Hospitals for supplying synovial tissue; and Drs. Edward Schwarzand Richard Phipps for their insightful discussion of the manuscript.

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Figure 8. Multigene analysis of FLS mRNA in unstimulated and IL-10treated (10 ng/ml) cultures. Unstimulated cells constitutively produce IL-1ß, IL-6, collagenase, and HLA-DRß. IL-10 decreases proinflammatorycytokine, collagenase, and HLA-DRß gene expression.




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