ARTHRITIS & RHEUMATISMVol. 43, No. 2, February 2000, pp 311–319© 2000, American College of Rheumatology

CYCLOSPORIN A PREVENTS THE HISTOLOGIC DAMAGEOF ANTIGEN ARTHRITIS WITHOUT INDUCING FIBROSIS

MARIA J. BENITO, OLGA SANCHEZ-PERNAUTE, MARIA JOSE LOPEZ-ARMADA,PURIFICACION HERNANDEZ, ITZIAR PALACIOS,

JESUS EGIDO, and GABRIEL HERRERO-BEAUMONT

Objective. To study the effects of cyclosporin A(CSA) in a model of rheumatoid arthritis (RA) and theactivation of growth factors related to pannus develop-ment at the site of injury.

Methods. Antigen arthritis was induced in theknees of 14 New Zealand rabbits, and the animals wererandomized into 2 therapeutic groups: CSA at 10 mg/kg/day and CSA solvent. After 3 weeks of treatment, therabbits were killed, and synovial tissues were obtainedand compared with healthy specimens with regard tohistopathologic lesions, deposition of transforminggrowth factor b (TGFb) and collagens, and messengerRNA expression of platelet-derived growth factor B(PDGF-B) and TGFb. The effect of CSA on the expres-sion of TGFb and PDGF-B was also examined incultured synovial cells.

Results. CSA administration alleviated the histo-logic damage and avoided the overdeposition of matrixelements in the injured tissue. It was also able tonormalize the enhanced expression of TGFb andPDGF-B observed in the untreated rabbits. Despite thismodulation found in vivo, CSA up-regulated in a dose-dependent manner the gene expression of both trophicfactors by healthy cultured synovial cells.

Conclusion. The present study shows that contin-uous administration of CSA prevents the developmentof chronic synovitis in an experimental model of RA. Asreported in other cell types, CSA promoted TGFb

transcription by synovial cells in vitro, but failed todisplay a profibrogenic effect in the inflamed envi-ronment.

Rheumatoid arthritis (RA) is a disease of un-known etiology characterized by synovial inflammationand joint destruction. Tissue infiltration by lymphocytesdetermines the inflammatory response that arises in therheumatoid synovium and leads to an enhancement ofthe number and activation of local cells (1). The patternof lymphokine production in this disease suggests a Th1cell–dependent process (2). Hence, several drugs havebeen employed in an effort to shut down the cascade ofinflammatory events that follow the interaction of macro-phages and T cells. Cyclosporin A (CSA) was introducedfor the treatment of RA because of its ability to inhibita variety of T cell responses, such as the antigen-inducedproduction of interleukin-2 (IL-2), IL-3, interferon-g(IFNg), and tumor necrosis factor a (3).

With the progression of RA, the synovial mem-brane grows, invading cartilage and bone, and at thisstage, macrophages and synovial fibroblasts play animportant pathogenic role regardless of T cell stimula-tion (4). Interestingly, there is a high discordance be-tween inflammatory activity and joint destruction (5),and effective antiinflammatory agents often have littleor no influence on the progression of the disease.

Together with cellular proliferation, develop-ment of the invading phenotype of the synovium isdefined by an accumulation of matrix elements. Extra-cellular matrix (ECM) is constituted by a meshwork ofpeptide domains and glycoside residues, which maintaina dynamic interaction and regulate cellular metabolism(6). Many cytokines bind to this substrate, which there-fore serves as a sequestration site from which growthfactor stores can be concentrated and promptly released(7). Transforming growth factor b (TGFb) and platelet-derived growth factor (PDGF) are two key cytokines in

Supported by a grant from Novartis, Madrid, Spain. Dr.Palacios’ work was supported by the Fundacion Inigo Alvarez deToledo.

Maria J. Benito, BSc, Olga Sanchez-Pernaute, MD, MariaJose Lopez-Armada, PhD, Purificacion Hernandez, BSc, Itziar Pala-cios, PhD, Jesus Egido, MD, Gabriel Herrero-Beaumont, MD: Fun-dacion Jimenez Dıaz, and Universidad Autonoma, Madrid, Spain.

Address reprint requests to Gabriel Herrero-Beaumont, MD,Fundacion Jimenez Dıaz, Avenida Reyes Catolicos 2, 28040 Madrid,Spain.

Submitted for publication July 13, 1999; accepted in revisedform September 23, 1999.

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the acquisition of an aggressive phenotype by the syno-vium and are detached from their binding to ECMduring inflammation by the action of local proteases (8).

TGFb is produced by macrophages, lymphocytes,and synovial fibroblasts. It is the main inductor of ECMaccumulation in inflammatory conditions (9). TGFbshows an immunosuppressive activity since it blocksmaturation of natural killer cells, but on the whole, it isconsidered deleterious in RA and related to pannusdevelopment (10). TGFb is also a strong stimulus for theprogression of cell cycle in fibroblasts, albeit the mostpotent mitogenic cytokines for synovial cells are by farthe PDGFs (11). Biologically active PDGF is a dimercomposed of any of the 3 possible combinations of the 2chains, A and B, of the molecule. PDGFs may beproduced by the recruited monocytes arriving from thecirculation or by local synoviocytes, and they stimulateDNA synthesis and proliferation of human synovialfibroblasts and endothelial cells (12).

Besides inhibition of IL-2 and IFNg, CSA alsopromotes the expression of TGFb1 by T lymphocytes aswell as by several nonimmune cells. It is accepted thatTGFb has a central role in the detrimental effects ofCSA in human disease (13). In fact, gingival and renaltoxicity of CSA consists of a fibrotic overgrowth andscarring of the involved tissues. Recently, it has evenbeen suggested that CSA may favor tumor spreadingthrough the generation of TGFb in several epithelialtumor cell lines (14).

Since increased production of TGFb by CSAwithin the inflamed synovium could favor pannus for-mation, we set up the present study to explore the effectsof CSA in an experimental model of RA. With this aim,we examined deposition of ECM and the synovial ex-pression of TGFb and PDGF-B, along with the histo-logic features of joint damage.

MATERIALS AND METHODS

Pharmacologic studies. Based on previous trials, weselected a CSA dosage of 10 mg/kg/day and the CSA solventethanol–oil (1:7) to carry out this study (15,16). Three healthyNew Zealand (NZ) rabbits were treated for 11 days with thisdosage to confirm that it yielded therapeutic plasma levels.Concentration of the drug was determined by a monoclonalfluorescence polarization immunoassay using the TDx opera-tion system (Abbott, North Chicago, IL) according to themanufacturer’s instructions.

Experimental design for antigen-induced arthritis.Figure 1 outlines the induction of antigen arthritis. Briefly, 14adult NZ rabbits with an initial weight of 2.5–3.0 kg wereimmunized with intradermal injections of 4 mg/ml of ovalbu-min (OVA; Sigma, St. Louis, MO) in Freund’s completeadjuvant (Difco, Detroit, MI). Animals were reimmunized 2

weeks later, as previously described (17). Five days after thesecond immunization, the antigen (5 mg of OVA dissolved in1 ml of sterile saline) was injected into the rabbits’ knees.

Animals were then randomized into 2 groups of 7 to betreated with either 10 mg/kg/day of CSA intramuscularly orwith CSA vehicle (ethanol–oil 1:7). Treatment was initiated 24hours before the joint injection and was continued for 22 days,at which time, the animals were killed and their infrapatellarsynovial membranes were removed. One membrane from eachrabbit was frozen at 270°C and used for RNA studies, and theother was fixed in formalin and used for morphologic evalua-tion. Seven healthy animals matched for age and weight wereused as controls in the studies.

Experimental procedures were reviewed and approvedby the Institutional Care Committee in accordance with inter-national guidelines.

Synovial cell cultures. Synovial cells were obtainedfrom the infrapatellar synovial membranes of healthy NZrabbits and characterized as previously described (18). Briefly,synovial explants were exposed to enzymatic digestion with1.25 mg/ml of trypsin for 1 hour with agitation (Boehringer,Mannheim, Germany). Collected cells were resuspended inRPMI 1640 medium (Gibco BRL, Paisley, UK) enriched with10% fetal calf serum (BioWhittaker, Walkersville, MD) andsupplemented with 2 mM glutamine (Gibco), 100 units/mlpenicillin, and 100 mg/ml streptomycin. Cells were plated inpetri dishes (Costar, Cambridge, MA), cultured at 37°C in 5%CO2, and used between the fourth and sixth passages.

Synovial cells were also collected from arthritic rabbitsfor in vitro studies. Animals were immunized with OVA asdescribed, and synovial explants were removed 7 days afterintraarticular injection of the antigen. Isolation and cultureprocedures were similar to those of healthy synovial cells, butthe arthritic cells were used at the second passage toprevent the loss of their phenotype.

Synovial histopathologic studies. Synovial membraneswere fixed in 10% phosphate buffered formalin for 16 hours,dehydrated in graded ethanols, and embedded in paraffin.Tissues were cut at 7 mm and stained with hematoxylin andeosin to characterize the injury at the synovial lining, sublining,

Figure 1. Experimental design for the arthritis induced by ovalbumin(OVA) in New Zealand white rabbits. Fourteen rabbits were immu-nized and treated with cyclosporin A (CsA; n 5 7) or CsA vehicle (n 57). Seven healthy rabbits matched for age and weight were used ascontrols.

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and vessels, according to a modification of a previously de-scribed method (19).

The severity of the lesions at each compartment wasscored according to a semiquantitative 5-point scale, and thesum of the scores was the “inflammation score,” which re-flected global tissue damage. Masson’s trichrome was used toassess deposition of ECM proteins in the synovial specimens.Findings were graded semiquantitatively (from 0 5 minimal to3 5 maximal). In both studies, 2 observers (MJB and OS-P)who were blinded to the experimental group evaluated thetissues.

Detection of TGFb, collagens, and macrophages insynovial tissues. Paraffin-embedded synovial tissues were sec-tioned at 7 mm and mounted on poly-L-lysine–precoatedcrystal slides. After deparaffinization, samples were rehy-drated, and endogenous peroxidase was inactivated by a 30-minute immersion in 1% hydrogen peroxide–methanol. Forthe detection of TGFb, slides were incubated with a blockingsolution (10% bovine serum albumin [BSA] and 4% horseserum in phosphate buffered saline [PBS]) for 20 minutes atroom temperature. Then, sheep anti-human TGFb (R&DSystems, Abingdon, UK) was added (1:100) and incubationscontinued overnight at 4°C. Donkey anti-sheep biotinylatedIgG (Amersham, Buckinghamshire, UK) diluted 1:200 wasused as secondary antibody. Color development was obtainedby peroxidase-labeled ABC complex (Dako, Carpinteria, CA)and diaminobenzidine (DAB).

Immunoreactivity to collagen types I and III (CI andCIII) was shown after digestion of the tissues with 0.1% trypsin(in 0.05M Tris HCl with 0.1% CaCl2, pH 7.8) for 30 minutes at37°C to retrieve the target antigen. Polyclonal antibodiesagainst human CI (1:10) and CIII (1:20) raised in goat (SeraLab, Crowley Down, UK) were used, and a peroxidase-labeleddonkey anti-goat IgG (1:200; The Binding Site, Birmingham,UK) served as secondary antibody. The monoclonal antibodyraised in mouse Ram-11 (Dako) was employed to detectmacrophages. After treatment with hydrogen peroxide–methanol, slides were incubated with 6% BSA and 2% sheepserum in PBS for 20 minutes, and the specific antibody (75mg/ml) was added. The second antibody, a peroxidase-labeledsheep anti-mouse IgG (Amersham) was diluted 1:200 for a30-minute incubation at room temperature.

DAB was used as chromogen, and tissues were coun-terstained with methyl green. The density of macrophages wasassessed by blinded scoring using a 0–4 scale.

RNA isolation, reverse transcription (RT), and poly-merase chain reaction (PCR) analysis. RNA was extracted bythe acid guanidine thiocyanate–phenol–chloroform method(20) and quantified by absorbance at 260 nm. Ten microgramsof RNA was denatured and electrophoresed in a 1% agarose–formaldehyde gel to verify RNA quality. Then, 50 ng of RNAwas used in the RT and semiquantitative PCR, according tothe manufacturer’s instructions (Promega, Madison, WI).

PCR analyses for TGFb, PDGF-B, and the housekeep-ing gene GAPDH were conducted under the following condi-tions: 1 minute at 94°C to denature the double-stranded DNA,2 minutes at 58°C/61°C/54°C, respectively, to allow annealingof the primers, and 2 minutes at 68°C for primer extension.

The primers used were as follows: antisense 59-TCA-TGT-TGG-ACA-GCT-GCT-TCC-39 and sense 59-TCC-GCA-AGG-ACC-TCG-GCT-GG-39 for TGFb that yielded a prod-uct of 243 bp; antisense 59-CCT-CCA-GGG-TCA-CTG-

TGG-39 and sense 59-CTG-AGG-GGG-ATC-CCA-TTC-39for PDGF-B (462 bp), and antisense 59-ATA-CTG-TTA-CTT-ATA-CCG-ATG-39 and sense 59-AAT-GCA-TCC-TGC-ACC-ACC-AA-39 for GAPDH (515 bp). Amplifications weredone for 15, 20, 25, 30, and 35 cycles to establish the linearityof the reaction and to determine the appropriate number ofcycles (25 for TGFb, 30 for PDGF-B, and 20 for GAPDH).

The DNA products were analyzed on 4% polyacryl-amide/urea gels and exposed to x-ray film. Autoradiogramswere scanned with the ImageQuant densitometer (MolecularDynamics, Sunnyvale, CA). Data were standardized to thehousekeeping gene GAPDH, and results expressed as thepercentage of basal gene expression (arbitrary densitometricunits).

In situ hybridization of PDGF-B and TGFb messengerRNA (mRNA). Specific probes were obtained from RT-PCRproducts inserted in the P-gem T Easy Vector and transfectedto Escherichia coli according to the manufacturer’s instructions(Promega) and then labeled with digoxigenin.

Paraffin-embedded synovial tissues were fixed in 1.5%paraformaldehyde–glutaraldehyde and immersed in 5 mMlevamisole for 30 minutes. Then, tissues were washed with 23saline–sodium citrate (13 SSC 5 150 mM NaCl, 15 mMsodium citrate, pH 7.0), digested with 5 mg/ml pepsin (Sigma)in 0.1N HCl, pH 1.5, for 15 minutes at 37°C, and dehydrated.Air-dried sections were covered with 20 ml of hybridizationsolution (43 SSC, 0.23 phosphates, and 23 Denhardt’s solu-tion) containing vanadyl ribonucleoside complex at a finalconcentration of 19.35 mM, 0.1 mg/ml of transfer RNA, and0.2 mg/ml of salmon sperm DNA. Dextran sulfate (20%) informamide and the sense or the antisense probe labeled withdigoxigenin were added to the solution. Hybridizations werecarried out overnight at 42°C in humid chambers undercoverslip protection.

Each section was washed sequentially in 23 SSC, 13SSC, and 0.13 SSC and incubated with an antidigoxigenin IgGlabeled with alkaline phosphatase (Boehringer). Color wasdeveloped with BCIP (X-phosphate) and nitroblue tetrazoliumsalt, and tissues were mounted in glycerol.

Statistical analysis. Statistical analysis was applied tothe semiquantitative evaluations. Concordance of the 2 obser-vations for each categorical variable was assessed by the kappaindex. The evaluations from observer 1 were used to developthe descriptive statistics, analysis of variance, and comparisonsof means by independent t-test. Data are expressed as themean 6 SD, and differences are considered significantat P , 0.05.

RESULTS

Plasma levels of CSA. At the beginning of thisstudy, we determined plasma concentrations of CSA inhealthy rabbits after 11 days of parenteral administra-tion of the drug. The 10 mg/kg/day dosage of CSAyielded a plasma concentration of 184 6 32 ng/ml(mean 6 SD), which is within the therapeutic rangeestablished in humans (100–250 ng/ml) (21,22).

Histologic lesions of the synovial membrane.After 3 weeks of treatment, rabbits were killed and

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macroscopic examination revealed that all untreatedanimals had developed the typical lesions of chronicarthritis (17,23).

Figure 2 shows the microscopic features of syno-vial tissues after staining with hematoxylin and eosin.Briefly, these alterations can be grouped into 3 differentevents: proliferation of the synovial lining cells, infiltra-tion of the sublining by mononuclear cells frequentlyorganized in aggregates, as in rheumatoid disease, andincreased vascularization of the hypertrophic tissues.The evaluation revealed a significant diminution of thelesions in the animals which received CSA, as reflectedin the inflammation score: 7.1 6 1.7 (mean 6 SD) inuntreated animals versus 3.7 6 2.7 in CSA-treatedanimals (P , 0.05). Figure 2D shows the individualscores for each compartment.

Antigen arthritis resembles RA, not only in theinflammatory cellular events, but also in ECM accumu-lation, which leads to a fibrotic overgrowth (24). Mas-son’s trichrome staining was employed to explore theability of CSA to down-regulate matrix deposition at thesynovial tissues (Figure 3). The arthritic animals exhib-ited an extended scarring pattern underneath the lining

layer and surrounding inflammatory aggregates. CSAtreatment resulted in a significant reduction of ECMaccumulation, as shown by the semiquantitative score:3.0 6 1.2 (mean 6 SD) in untreated rabbits versus 1.6 61.0 in CSA-treated rabbits (P , 0.05).

TGFb production and deposition of collagens inthe arthritic synovial membranes. It is well establishedthat CI and CIII are the main ECM proteins involved infibrosis associated with chronic inflammation (25,26)and that TGFb, which is activated by local proteases,plays a role. Using immunoperoxidase techniques, weobserved an important reduction in the amount of thisgrowth factor in the synovium of CSA-treated animalscompared with untreated animals. While the lattershowed a generalized immunoreactivity to TGFbthroughout the tissue, CSA-treated animals exhibitedTGFb only at the surface, similar to healthy controls(Figures 4A and C). Figures 4D–F and Figures 4G–Iillustrate the changes observed in the distribution pat-terns of CI and CIII, respectively.

Untreated animals displayed a sizable enhance-ment in the signal for CI and CIII. Expression of theECM components was particularly prominent around

Figure 2. Representative photomicrographs of hematoxylin and eosin–stained sections ofsynovial membranes from A, healthy, B, untreated, and C, cyclosporin A (CsA)–treated NewZealand white rabbits. While untreated animals exhibited a massive hyperplasia and recruit-ment of inflammatory cells, CsA administration alleviated the leukocyte infiltration andreduced the proliferation of synovial cells. (Magnification 3 100.) D, Semiquantitative scores oflining, infiltrated cells, and vessels in the different study groups. Significant differences insublining infiltration of leukocytes were found between untreated and CsA-treated animals(p 5 P , 0.05).

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vessels and underneath the lining layer. CSA adminis-tration reduced the accumulation of both collagenswithin the tissues. As shown in Figure 4, healthy synovialtissues had a low immunoreactivity to these ECM pro-

teins, particularly those localized to the deeper areas andorganized in connective tracts.

Messenger RNA expression of TGFb and PDGFat the synovium. Along with the activation of presynthe-

Figure 3. Masson’s trichrome–stained sections, demonstrating the alterations in the distribution of extracellular matrix (ECM)proteins produced in the experimental disease. A, In healthy tissues, there was a low staining (green) at the interstitium. B,Untreated animals showed a wide distribution of ECM elements along the membrane. C, Cyclosporin A–treated animals displayeda similar pattern to that of healthy controls, significantly down-regulating the overdeposition of ECM proteins noted in theuntreated animals (P , 0.05). (Magnification 3 200.)

Figure 4. Immunolocalization of A–C, transforming growth factor b, D–F, type I collagen, and G–I, type III collagen in the synovialmembranes of healthy (left), untreated (middle), and cyclosporin A–treated (right) New Zealand white rabbits. (Hematoxylincounterstained; magnification 3 200 in A–C; 3 100 in D–I.)

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sized growth factors, there is an enhanced transcriptionof these peptides during inflammation that seems to bea determinant of the hypertrophic transformation of thetarget tissue. By RT-PCR techniques, we detected aconsiderable rise in TGFb and PDGF-B mRNA expres-sion (20% and 35%, respectively) in untreated animals,while CSA-treated rabbits did not show this up-regulation (Figure 5).

With the aid of in situ hybridization techniques,we could look at the distribution of growth factorexpression in the inflamed tissues (Figure 6). TGFbexpression was found at the lining layer and in theinfiltrated aggregates, the same areas as TGFb protein.There was also a strong hybridization signal forPDGF-B, related to the lining layer, sublining cells, andvessel walls, where it comprised endothelium and adven-titia, as has been described in other tissues (27).

Staining of macrophages in synovial tissues.Macrophages are possibly the ideal target cells in thetreatment of chronic inflammation (26) and a mainsource of growth factors at the injured site. Since CSAinhibits IFNg production by T lymphocytes, its beneficialeffect in inflammation could be due to an impairment ofmacrophage activation (28). We investigated the density

of macrophages within the synovial samples by thebinding of Ram-11 (Figure 7). In most of the healthyspecimens, stained cells were seldom found in the innertissue, and they appeared in a low percentage at thelining (mean 6 SD score 0.14 6 0.38). The untreatedgroup of rabbits with antigen arthritis showed a predom-inance of macrophages among the cells of the sublining,in areas where granulation tissue was arising. Some ofthem had the typical morphology of foam cells and werereplacing local adipocytes of the normal infrapatellarsynovium (score 3.14 6 0.90). The proportion of liningcells with a macrophage phenotype was also remarkablyincreased in these animals.

Samples from the CSA-treated group had a no-table diminution in the amount of macrophages com-pared with samples from the untreated group (score2.0 6 0.82; P , 0.05), particularly evident at thesublining. Interestingly, the distribution pattern ofRam-11 resembled that of PDGF mRNA, as shown inFigures 7D and E. This suggests that macrophages areinvolved in the overproduction of PDGF-B found inantigen arthritis.

TGFb expression by cultured synovial cells afterCSA stimulation. Since CSA has been shown to enhanceTGFb transcription in several cell types, we next usedPCR techniques to investigate the effect of the drug onthe production of TGFb by B synoviocytes. The cellscultured in petri dishes were grown to subconfluence,starved, and exposed to increasing concentrations ofCSA (1–1,000 ng/ml) for 8 hours. At that time, CSAelicited a dose-related increase in mRNA levels ofTGFb that were 8-fold higher than in unstimulated cellsat the 1,000 ng/ml concentration (Figure 8A).

Cells under chronic stimulation, such as synovialcells from rheumatoid tissues, usually have a differentresponse to stimuli than healthy cells. Thus, we nextexplored the gene expression of TGFb in the cellsobtained from rabbits with antigen arthritis. The ar-thritic cells exhibited a high mRNA expression of TGFbthat was not further increased when cells were treatedwith CSA. In fact, at the lowest concentrations tested(1–10 ng/ml CSA), the drug abrogated this growth factoroverexpression (Figure 8B).

DISCUSSIONThe selection of antigen arthritis for these studies

was based on its resemblance to RA, not only withregard to cellular activation, but also to fibrotic over-growth of the synovium, reflecting pannus formation.Given the high deposition of ECM elements at thesynovial pannus, we expected TGFb and PDGF-B,

Figure 5. Gene expression of transforming growth factor b (TGF-b)and platelet-derived growth factor B (PDGF) in the synovial tissueswas determined by reverse transcription–polymerase chain reaction.GAPDH was used as a housekeeping gene to normalize the messengerRNA (mRNA) levels of both growth factors. The graph shows mRNAexpression as a percentage of the levels found in healthy tissues afterdensitometric analysis. The animals that remained untreated exhibitedan increase in TGFb and PDGF mRNA that was not found in thecyclosporin A (CsA)–treated group.

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perhaps the most important growth factors in the acqui-sition of the invasive phenotype in RA, to be activated inthis experimental model of RA. Indeed, we observed anup-regulation of the metabolism of these peptides in thearthritic tissues, showing a good correlation with the

histopathologic lesions, as determined by hematoxylinand eosin and Masson’s trichrome staining. In view ofour results, the expression of TGFb and PDGF-B withinthe inflamed tissue could prove to be a fine marker ofdisease evolution and response to therapy.

Figure 6. Representative photomicrographs of sections of synovial membranes from A and E, healthy, B and F, untreated, and C andG, cyclosporin A–treated New Zealand white rabbits after in situ hybridization of specific antisense probes for transforming growthfactor b (TGFb) and platelet-derived growth factor B (PDGF-B). D and H, Internal controls in which the arthritic tissues werehybridized with the sense probes of TGFb and PDGF-B mRNA, respectively. (Magnification 3 200 in A–D; 3 100 in E–H.)

Figure 7. Representative photomicrographs of sections of synovial membranes from A,healthy, B, untreated, and C, cyclosporin A (CSA)–treated New Zealand white rabbits, showingmacrophage immunodetection. CSA treatment resulted in a significant clearance of these cellsfrom the arthritic joint (P , 0.05). Note the parallel distribution of Ram-11 immunoreactivity(D) and platelet-derived growth factor B mRNA (E) at the subintimal level in tissues from theuntreated arthritic rabbits. (Methyl green counterstained; magnification 3 200.)

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The design of our study was based on the ac-knowledged role of CSA in the development of fibrosis(29,30). On one hand, CSA inhibits IL-2 synthesis andsubsequent maturation of T4 lymphocytes, and it alsoalters the activation of macrophages because of a down-regulation of IFNg production. It is widely recognizedthat interaction of antigen-presenting cells and lympho-cytes is decisive in the development of RA. On the otherhand, stimulation of TGFb in different systems is relatedto angiogenesis, fibroblast proliferation and activation,loosening from matrix attachment and spreading ofdifferent cells, and most of all, ECM overdeposition andscarring (31). However, in our study, CSA did not elicitTGFb or PDGF-B expression in vivo and it avoidedECM accumulation and lining hyperplasia.

A possible source of TGFb during inflammationis the Th3 subset of lymphocytes (32). There is, none-theless, considerable evidence showing a selective migra-tion of Th1 cells to the inflamed synovial membrane inRA (33), this being a possible explanation for theabsence of fibrotic transformation of the synovial spec-imens under CSA treatment.

Synovial fibroblasts could also account for localproduction of growth factors. There are no previousstudies of the kinetics of CSA in these cells, andextrapolation is complex because CSA disposes of sev-eral reservoirs in the organism, which can alter itsdistribution to the tissues. At the cellular level, theplasma membrane and CSA cytoplasmic transporters, orcyclophilins, may bind CSA for a long time. These

binding proteins are differentially regulated among in-dividuals, tissues, and even cells within a tissue (34). Asa consequence, CSA evokes variable responses, partiallydepending on the differences in cyclophilin expression.Thus, it could happen that CSA, did not raise a fibro-genic response by synoviocytes (35) as it did in other celltypes. Our in vitro experiments, however, showed thatCSA promotes the expression of TGFb and PDGF-B bysynovial fibroblasts. CSA acts at the transcription level,and in other cell types, there is a fine correlationbetween mRNA expression, synthesis, and activity of thecytokine after CSA stimulation (36). Therefore, al-though we did not perform specific experiments, we canassume that the same increase in TGFb productionfollows transcription in synovial cells.

Finally, macrophages are probably the most im-portant origin of TGFb and PDGF-B (37) within theinflamed joint and could be a target for CSA in arthritis.In fact, CSA not only alters cytokine production andantigen presentation by macrophages, but the migrationof these cells may also become impaired by the drug, asreported elsewhere (38). In our study, we were able todemonstrate a clearance of macrophages at the inflam-matory aggregates of the sublining in CSA-treated rab-bits. In this sense, the use of a prophylactic approachcould be the determinant of the abolition of TGFb andPDGF-B up-regulation in vivo, and it would be interest-ing to study the effects of CSA on established disease.With that aim, we performed the experiments in arthriticsynoviocytes, where the enhancement in TGFb gene

Figure 8. Transforming growth factor b (TGF-b) mRNA expression was investigated byreverse transcription–polymerase chain reaction in A, healthy and B, untreated arthriticsynovial cells after an 8-hour incubation with cyclosporin A (CsA; 1–1,000 ng/ml). Shownare representative blots of 3 experiments performed. The mRNA concentration wascalculated after correction against GAPDH levels and is represented as the percentage ofexpression of TGFb in unstimulated cells. While CsA elicited a dose-dependent inductionof TGFb gene expression in healthy synovial cells, the arthritic synovial cells showed a highbasal expression of TGFb that was not up-regulated by CsA treatment.

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expression was not evident. The data suggest that thereare local environmental factors that may interfere withcell behavior and explain why TGFb does not mediatethe actions of CSA in inflamed tissues.

In summary, we performed an in-depth study ofthe pathophysiology of antigen arthritis, an experimentalmodel of RA, and we provide new evidence of the roleof growth factors in tissue damage. This study alsodemonstrates the efficacy of the prophylactic employ-ment of CSA in antigen arthritis, in which the drugavoids fibrotic transformation of the synovial tissue.

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