Effects of Leflunomide on Hyaluronan (HAS): NF- κB-Independent Suppression of IL-1-Induced HAS1 Transcription by Leflunomide This information is current as of September 28, 2021. Karl M. Stuhlmeier J Immunol 2005; 174:7376-7382; ; doi: 10.4049/jimmunol.174.11.7376 http://www.jimmunol.org/content/174/11/7376 Downloaded from

References This article cites 59 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/174/11/7376.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Effects of Leflunomide on Hyaluronan Synthases (HAS): NF-␬B-Independent Suppression of IL-1-Induced HAS1 Transcription by Leflunomide1

Karl M. Stuhlmeier2

Despite evidence that points to unfettered (HA) production as a culprit in the progression of rheumatic disorders, little is known about differences in regulation and biological functions of the three hyaluronan (HAS) . Testing the effects of drugs with proven anti-inflammatory effects could help to clarify biological functions of these genes. In this study, we demonstrate that leflunomide suppresses HA release in fibroblast-like synoviocytes (FLS) in a dose-dependent manner. We further demonstrate that leflunomide suppresses HA synthase activity, as determined by 14C-glucuronic acid incorporation assays. Ad- ditional experiments revealed that in FLS, leflunomide specifically blocked the induction of HAS1. HAS2 and HAS3, genes that Downloaded from are, in contrast to HAS1, constitutively expressed in FLS, are not significantly affected. Leflunomide can function as a NF-␬B inhibitor. However, EMSA experiments demonstrate that at the concentrations used, leflunomide neither interferes with IL-1␤- nor with PMA-induced NF-␬B translocation. Furthermore, reconstituting the pyrimidine synthase pathway did not lead to the restoration of IL-1␤-induced HAS1 activation. More importantly, two tyrosine kinase inhibitors mimicked the effect of leflunomide in that both blocked IL-1␤-induced HAS1 activation without affecting HAS2 or HAS3. These data point at HAS1 activation as the

possible cause for unfettered HA production in and might explain, at least in part, the beneficial effects of http://www.jimmunol.org/ leflunomide treatment. These findings also support the concept that IL-1␤-induced HAS1 activation depends on the activation of tyrosine kinases, and indicate that leflunomide blocks HA release by suppressing tyrosine kinases rather than through inhibition of NF-␬B translocation. The Journal of Immunology, 2005, 174: 7376–7382.

heumatoid arthritis (RA),3 a disorder of unknown etiol- of HA possess a series of unwanted properties such as acting as ogy, affects large parts of the population (1–4). This de- chemoattractants, inducing blood vessel growth, as well as acti- R bilitating disease is characterized by chronic inflamma- vating and inducing the release of proinflammatory molecules in tion of affected joints caused by infiltrating cells. Structural leukocytes and endothelial cells (10–15). That detection of in- damage is caused by a series of well-described mediators of in- creased levels of HA in human synovium effusion serves as a sen- by guest on September 28, 2021 flammation. Among the known participants in inflammatory pro- sitive indicator of altered connective tissue cell function has been cesses are adhesion molecules, cytokines, and chemokines. Al- recognized for some time (16). Furthermore, while in healthy though the consequences of chronic inflammation are well joints only a fine layer of HA covers and protects joint surfaces, described, very little is known about early events that cause, for RA is characterized by uncontrolled HA production, often leading example, unwanted migration of leukocytes into joints. to enormous amounts of HA in affected joints. Therefore, signif- Controlled hyaluronic acid (HA) production/release is undoubt- icantly elevated levels of HA can be detected even in the serum of edly essential for many physiological mechanisms, for example, RA patients, and elevated plasma HA levels correlate positively the proper functioning of joints. Nevertheless, unfettered HA pro- with destruction of involved joints (11). As a result, it has been duction results in many detrimental effects and might directly as suggested to use HA serum levels as indicators of disease progres- well as indirectly contribute to the progression of RA. This hy- sion (17). In strong support of the hypothesis that HA is a key pothesis is supported by a series of findings, demonstrating for player in RA, Wang and Roehrl (18) showed in an animal model example that increased HA synthesis is nearly always associated that within 50 days of weekly HA injections, animals became with inflammatory reactions, irrespective of its cause (5–9). In ad- chronically sick, exhibiting on-and-off RA symptoms for months. dition, as has been demonstrated many times, degradation products The animals in this study also developed all classical pathological symptoms of RA, such as swollen joints, edema, large numbers of CD4ϩ cells, and infiltrating macrophages (18). Ludwig Boltzmann Institute for Rheumatology and Balneology, Vienna, Austria HA can be synthesized by three distinct that are the Received for publication October 15, 2004. Accepted for publication March 21, 2005. products of transcription and translation of the genes hyaluronan The costs of publication of this article were defrayed in part by the payment of page synthase (HAS) 1, HAS2, and HAS3 (19). Earlier, we tested the charges. This article must therefore be hereby marked advertisement in accordance effects of a series of proinflammatory cytokines on the expression with 18 U.S.C. Section 1734 solely to indicate this fact. of the three HAS genes in fibroblast-like synoviocytes (FLS) (20). 1 This work was supported in part by grants from the City of Vienna; the Austrian Ministry of Social Security and Generations; the Austrian Ministry of Education, These studies revealed that in cultured FLS, the genes HAS2 and Science, and Culture; and the Austrian National Bank. HAS3 are constitutively expressed, while mRNA for HAS1 is very 2 Address correspondence and reprint requests to Dr. Karl M. Stuhlmeier, Ludwig low or undetectable. More importantly, stimulation with proin- Boltzmann Institute for Rheumatology and Balneology, Kurbadstrasse 10, 1100 Vi- flammatory cytokines revealed that in FLS HAS1 mRNA is readily enna, Austria. E-mail address: [email protected] inducible, resulting in a manifold increase in HAS1 mRNA as well 3 Abbreviations used in this paper: RA, rheumatoid arthritis; CRE, cAMP-responsive element-like; FLS, fibroblast-like synoviocyte; HA, hyaluronic acid/hyaluronan; as in significantly elevated levels of HA as determined by ELISA. HAS, ; LB, lysis buffer. Interestingly, very little or no changes were noticed monitoring

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 7377

mRNA levels of HAS2 and HAS3 in such experiments. We con- HA measurements cluded from these experiments that HAS1 may be the in FLS Aliquots of culture medium were removed at indicated time points, cen- whose unfettered activation by proinflammatory cytokines might trifuged (5 min at 2000 ϫ g), and tested for the presence of HA via a be involved in elevated HA levels associated with RA. procedure provided by Corgenix. OD values were used to calculate HA With the objective of expanding our understanding with re- levels using a third-order polynominal regression analysis performed with gard to the involvement of the three HAS genes and their reg- a universal assay calculation program (AssayZap; Biosoft). ulation in rheumatic diseases, we tested a series of drugs that In vitro HAS assay have been used successfully to treat various forms of rheumatic disorders. In this work, we report the results of experiments HAS activity was monitored using a modification of previously described treating FLS with leflunomide, a drug that has been shown to be methods (30). Briefly, FS, cultured in 15-cm tissue culture dishes, were washed, incubated in hypotonic lysis buffer (LB) (10 min), and harvested very effective not only in the treatment of rheumatic disorders into 1 ml of LB supplemented with aprotinin, leupeptin, and PMSF. A (21–24), but also as a remedy in preventing the rejection of Dounce homogenizer (pestle B) was used to disrupt cells. Nuclei were allografts as well as xenografts (25–27). pelleted by spinning tubes at 1,000 ϫ g for 4 min. Samples were centri- fuged at 16,000 ϫ g for 25 min to pellet membrane fragments. LB (50 ␮l) Materials and Methods with protease inhibitors was used to resuspend membrane pellets. An ali- quot, diluted in LB plus 1% SDS, was used for protein measurement using Reagents the bicinchoninic acid assay (Pierce). All of the above steps were per- If not stated otherwise, reagents were from Sigma-Aldrich. The erbstatin formed in the cold room using prechilled solutions. The in vitro HA syn- ␮ analog, 2,5-dihydroxymethylcinnamate and genistein, were from Calbio- thase assays using 25 g of cell membrane extract were assembled exactly 14 chem. UDP-[ C]glucuronic acid (418.3 mC/mmol) was from as described (30). After incubation for1hat37°C, the reaction was Downloaded from PerkinElmer. Oligonucleotides for EMSA experiments were from Pro- stopped by boiling, and the mixtures were further incubated with or without ␮ mega; HA-ELISA were from Corgenix. Abs for supershift experiments Streptomyces hyaluronate (40 turbidity-reducing units per 50- l al- were from Santa Cruz Biotechnology. iquot) at 37°C overnight and then treated with 200 mg/ml Pronase at 37°C for 5 h for deproteinization. Cell culture After boiling the samples in the presence of 1% SDS (w/v), mixtures were transferred to Microcon centrifugal filter devices that retained mole- Human fibroblast-like synoviocytes (FLS) were a gift from G. Partsch cules larger than 100,000 Da (Microcon YM-100; Millipore). Unincorpo- (Ludwig Boltzmann Institute, Vienna, Austria) (28) or were purchased rated [14C]glucuronic acid was removed by filtration (5 min at 5,000 ϫ g). http://www.jimmunol.org/ from Dominion Pharmakine. FLS were cultured, as previously described Subsequently, LB (200 ␮l) was added to the sample reservoir. Centrifu- (20). In brief, FLS were propagated in T75 tissue culture flasks or culture gation and resuspension of the retentate in LB were repeated three times to dishes (Iwaki, Funabashi) (15 cm diameter) in DMEM (Sigma-Aldrich) ensure complete removal of unincorporated [14C]glucuronic acid. After the supplemented with 10% heat-inactivated FBS (Sigma-Aldrich), L-glu- final spin, sample reservoirs were placed upside down in a new vial and tamine, and 50 U/ml penicillin/streptomycin. Medium was changed every centrifuged at 1,000 ϫ g to recover polysaccharides. Scintillation mixture 3 days. For experiments, FLS were detached using trypsin and transferred was added to determine radioactivity. [14C]Glucuronic acid incorporated to 6- or 24-well plates (Iwaki, Funabashi). For ELISA experiments, FLS into hyaluronan polymer was calculated from the Streptomyces hyaluroni- were cultured in 24-well plates, and for RT-PCR experiments in 6-well dase-sensitive radioactivity. plates, respectively.

RNA isolation and RT-PCR Quality control and data analysis by guest on September 28, 2021 RNA isolation, reverse transcription, and PCR were performed, as de- Special care was taken to terminate PCR in the log phase of amplification. scribed (20). Small aliquots of RNA were used to check the quality of RNA As demonstrated earlier (20, 31), a series of cycles are routinely tested to using agarose gel and ethidium bromide or Vistagreen (Molecular Probes) define optimal PCR conditions for a given gene. Viability of cells was for visualization. First-strand cDNA synthesis was performed exactly as confirmed by phase contrast microscopy and occasionally by staining cells described by the supplier of the RT-PCR kit (Amersham Biosciences) us- with trypan blue. ing 1 ␮g of RNA per reaction. Aliquots were used for PCR. A Techne Agarose gels were stained with ethidium bromide and scanned on a cycler (Techgene) and an Eppendorf cycler (Eppendorf) were used for PCR Fluorimager 595 (Amersham Biosciences). Data were analyzed and quan- under the following standard conditions: initial denaturation, 4 min at titated using ImageQuant Software (Amersham Biosciences). mRNA for 94°C; annealing, 55°C or 62°C (HAS3); amplification, 20 s at 72°C; de- GAPDH or actin or both were used as controls for RT-PCR, and scanner naturation, 20 s at 94°C; 21–35 cycles, followed by final extension for 10 readings were used to recalculate PCR data. min at 72°C. Care was taken to work out exact PCR conditions to ensure that the amplification reaction was stopped in the log phase of amplifica- tion. As control for equal usage of mRNA, either actin or GAPDH or both Results were used. IL-1␤-induced HA release is blocked by leflunomide Primers were from MWG Biotec and were dissolved at a concentration of 100 pmol/␮l in Tris-EDTA. The sequence of the primers for HAS1, FLS are cells that are able to produce and release considerable HAS2, HAS3, GAPDH, and actin mRNA as well as PCR conditions were amounts of HA (12, 14, 32, 33). In this study, we demonstrate that published before (20). Aliquots of PCR were separated on agarose gels. leflunomide is able to suppress induced HA release in FLS in a The specificity of PCR was confirmed by comparing the size of the am- dose-dependent manner. We tested the effects of leflunomide on plified fragment with the calculated length as well as by sequencing the basal and IL-1␤- as well as IL-1␣-induced HA release. IL-1 was PCR products. added, and cells were incubated for up to 24 h. Where indicated, EMSA FLS were treated with 5 and 50 ␮M leflunomide only, or were Nuclear extracts from FLS were prepared, as described (29). The double- pretreated with leflunomide 30–45 min before the addition of stranded oligonucleotides used in all experiments were end labeled using IL-1. Aliquots of cell culture supernatant were collected at times T4 polynucleotide kinase and [␥-32P]ATP. After labeling and purification ranging from 4 to 24 h. ␮ by chromatography, 5 g of nuclear extract was incubated with 100,000 Shown in Fig. 1 is one representative experiment in which HA cpm of labeled probe in the presence of 1.5 ␮g of poly(dI-dC) at room temperature for 20 min, followed by separation of this mixture on a 6% levels were quantitated by an HA-specific ELISA. Measurements polyacrylamide gel in Tris/glycine/EDTA buffer at pH 8.5. For specific were done in duplicates and demonstrate readily detectable HA competition, 7 pmol unlabeled NF-␬B oligonucleotides was included, and levels in culture medium of unstimulated FLS. In control experi- for nonspecific competition, 7 pmol double-stranded AP-1 oligonucleo- ments, HA concentrations were also measured in the complete tides and 7 pmol cAMP-responsive element-like (CRE) nucleotide were used. For supershift assays, 1 ␮l of the specific supershift Ab anti-NF-␬B culture medium (DMEM plus 10% FCS) used to culture FLS. HA p56 subunit mAb (Santa Cruz Biotechnology) was added to the nuclear levels in complete medium were negligibly low (Յ30 ng/ml). In extract simultaneously with the labeled probe. the experiment shown in Fig. 1, HA levels were quantitated after 7378 LEFLUNOMIDE PREVENTS HAS1 ACTIVATION

FIGURE 2. IL-1␤-induced HAS activity is blocked by leflunomide. FLS were treated with leflunomide and IL-1␤, respectively, as described in FIGURE 1. Leflunomide is a suppressor of induced HA release in FLS. Results. The dpm were monitored and reveal that leflunomide prevents Shown are the results of an ELISA experiment in which cells were left IL-1␤-induced HA synthase activity. The dpm in unstimulated FLS were untreated (column MEDIUM), stimulated with 5 ng/ml IL-1␤ (column 388 Ϯ SD 140; in IL-1␤-treated cells 1275 Ϯ SD 205; and in leflunomide- IL-1␤), treated with two concentrations of leflunomide (5 and 50 ␮M) treated cells 450 Ϯ SD 72. The cpm are given on the y-axis, and culture (column LEF 50 and LEF 5, respectively), or pretreated with indicated conditions are indicated on the x-axis. amounts of leflunomide, followed by stimulation with IL-1␤ (5 ng/ml). Consistently, HA levels in culture medium of FLS stimulated with IL-1␤ were twice as high as HA levels in medium of untreated cells. Leflunomide treatment blocked the IL-1␤ effect, but had no significant effect on basal Leflunomide selectively blocks HAS1 and has no significant HA release. Given on the y-axis are HA levels in ng/ml, and culture con- effect on HAS2 and HAS3 mRNA levels Downloaded from ditions are indicated on the x-axis. HA synthase assays measure net effects on HA synthase activity and cannot distinguish among the three HAS genes. To gain a better understanding of the mechanisms behind leflunomide effects on HA synthase, RT-PCR experiments were performed. FLS were 16 h. HA in wells containing unstimulated cells was found at con- left untreated, preincubated with 5 or 50 ␮M leflunomide for 30 centrations of 618 Ϯ 54 ng/ml. As shown in this figure, treating min before IL-1␤ (5 ng/ml) or PMA (2.5 ng/ml) treatment, or were http://www.jimmunol.org/ ␮ FLS with 5 M leflunomide had no effect on noninduced HA treated only with IL-1␤ and PMA, respectively. Experiments were ␮ release. Increasing the concentration of leflunomide to 50 M (col- terminated after 6 h; total RNA was isolated; and levels of HAS1, Ϯ umn LEF 50) led to an insignificant reduction of HA levels (618 HAS2, and HAS3 mRNA were evaluated by RT-PCR. Ϯ ␮ 54 ng/ml HA in unstimulated vs 499 47 ng/ml in 50 M le- Shown in Fig. 3, A and B, are representative experiments dem- ␤ flunomide-treated cells). Stimulating cells with IL-1 resulted in a onstrating that leflunomide suppresses IL-1␤-induced HAS1 Ͼ Ϯ 100% increase in measurable HA levels (1325 112 ng/ml). mRNA in a dose-dependent manner. Furthermore, as demonstrated ␮ More importantly, pretreating FLS with 50 M leflunomide com- in Fig. 3A, leflunomide does not exert any significant effects on ␤ Ϯ pletely abolished the effect of IL-1 (1325 112 ng/ml HA in mRNA levels of HAS2 or HAS3. HAS1 mRNA levels are low in

␤ Ϯ ␤ by guest on September 28, 2021 IL-1 -treated vs 521 54 ng/ml HA in leflunomide- and IL-1 - quiescent, unstimulated FLS (column Medium), stimulation with ␮ treated cells). Lowering the concentration of leflunomide to 5 M PMA (column PMA), as well as with IL-1␤ (column IL-1␤) led to ␤ still results in a reproducible and significant reduction of IL-1 - a significant increase of detectable HAS1 mRNA levels. In the Ϯ Ϯ induced HA release (1325 112 ng/ml vs 729 68 ng/ml). three independent experiments performed, leflunomide alone (col- umn Lef) had no significant effect on any of the HAS genes. More ␤ Leflunomide prevents IL-1 -activated HA synthase activity importantly, leflunomide dramatically affected induced HAS1 A series of enzymes are known that can be involved in controlling mRNA accumulation. As a comparison of the columns Lef ϩ levels of HA (34, 35). Activation of such hyaluronidases by le- PMA and Lef ϩ IL-1, with controls shown in columns PMA and flunomide might account for the observed effect. Therefore, we IL-1, demonstrates, treatment with leflunomide (50 ␮M) repeat- subsequently tested whether the effects on HA levels noted by edly led to a reduction of HAS1 mRNA levels by 75–100%. ELISA are a consequence of leflunomide-induced changes in HA Shown in Fig. 3 are gel scans and graphs resulting from quanti- degradation patterns, or due to leflunomide-induced changes of tating HAS1 mRNA levels by densitometry. HA synthase activity. An in vitro HA synthase assay was estab- Shown in Fig. 3B is a representative experiment that demon- lished, as published previously (30). FLS were grown to high den- strates the dose effects of leflunomide. Although in such experi- sity in 15-cm tissue culture dishes and treated with IL-1␤ (5 ng/ ments 5 ␮M leflunomide was sufficient to suppress IL-1␤-induced ml), with or without leflunomide (50 ␮M) for 8 h. Leflunomide HAS1 mRNA levels by 10–50%, increasing the amount of le- was added 30–40 min before the addition of IL-1␤. HA synthase flunomide to 50 ␮M resulted in 75–100% inhibition. mRNA for assays were performed, as described (20, 30). Hyaluronidase from the gene actin was coamplified and used to adjust HAS mRNA Streptomyces was used in control experiments that confirmed HA levels. specificity of the assay. Because IL-1␤ might also contribute to elevated HA levels by As shown in Fig. 2, leflunomide inhibits IL-1␤-induced HA syn- suppressing the synthesis of HA-degrading enzymes, we tested thase activity. Although in this particular experiment dpm in un- whether IL-1␤ affects the activation of HA-degrading enzymes. As treated cells (column Medium) were 388 Ϯ SD 140 (SD), there reported earlier (20), mRNA for HYAL1, HYAL2, as well as was a significant increase to 1275 Ϯ SD 205 in cells treated with PH-20 are readily detected in FLS. Of the remaining hyaluronidase IL-1␤. Exposure to leflunomide before the addition of IL-1␤ re- genes, HYAL3 is not measurable in synoviocytes, but readily de- sulted in a significant reduction to 450 Ϯ SD 72 dpm. Two inde- tectable in carcinoma cells used in our control experiments. pendent experiments were performed; shown are the results of one HYAL4 is a chondroitinase that has no activity against HA, and experiment done in duplicates. Decays per minute are given on the HYALP1 is a pseudogene. Shown in Fig. 3C are data demonstrat- y-axis, and culture conditions are indicated on the x-axis. Differ- ing that IL-1␤ treatment did not affect mRNA levels of the genes ences between IL-1␤-treated and leflunomide plus IL-1␤-treated encoding hyaluronidases in synoviocytes. Taken together, these cells are considered significant ( p ϭ 0.033). experiments indicate that the elevated HA levels resulting from The Journal of Immunology 7379

exposure to IL-1␤ are due to activation of the gene HAS1 rather than the down-regulation of HA-degrading enzymes

Leflunomide does not block IL-1␤-induced NF-␬B translocation in FLS NF-␬B is a transcription factor that plays an essential role in the activation of many proinflammatory genes (36, 37). IL-1␤ is known to induce activation and translocation of NF-␬B in several cell types. Furthermore, convincing evidence has been presented by others (38) indicating the potential of leflunomide as an inhib- itor of NF-␬B activation. We were interested in investigating whether inhibition of NF-␬B might account for leflunomide-me- diated inhibition on HAS1 mRNA. First, the effect of IL-1␤ on NF-␬B activation in FLS was tested. Cells were cultured in 10-cm tissue culture plates to high density and subsequently stimulated with IL-1␤. Nuclear extract prepara- tion and EMSA were performed, as described before (29). Shown in Fig. 4A is one of two experiments performed to determine the optimal point in time for subsequent experiments. As demonstrated Downloaded from in Fig. 4A, IL-1␤ (5 ng/ml)-treated FLS respond within a very short time period with the translocation of NF-␬B into the nucleus. Although 15 min of IL-1␤ treatment is sufficient to result in readily detectable NF-␬B binding to its consensus element in EMSA ex- periments, maximal levels of NF-␬B can be observed between 30

and 45 min. IL-1␤ concentrations as low as 0.1 ng/ml resulted in http://www.jimmunol.org/ readily detectable NF-␬B translocation; maximal activation was obtained at 3–5 ng/ml; and increasing IL-1␤ concentrations further did not result in any further significant increase (data not shown). On the left side of this EMSA figure, the label ␬B indicates the position of the NF-␬B/DNA complexes, and NS indicates the po- sition of non-␬B/DNA-binding protein complexes. Given on top of Fig. 4B are the culture conditions (minutes of IL-1␤ exposure). The label Free Pr. indicates the column in which nuclear protein extract was left out of the EMSA reaction mixture. by guest on September 28, 2021 Next, we tested whether leflunomide has the potential to affect IL-1␤-induced NF-␬B activation. FLS were treated with 5 and 50 ␮M leflunomide for 45 min; after that, IL-1␤ (5 ng/ml) was added. A representative EMSA experiment is shown in Fig. 4B.Asa comparison of the columns labeled Medium and IL-1, respec- tively, demonstrates, IL-1␤ treatment results in significantly higher levels of NF-␬B/DNA complexes. More importantly, neither pre- treating FLS with 5 ␮M nor with 50 ␮M leflunomide led to a

densitometry. Shown in the lower sector are sections of gels (mRNA for HAS1, HAS2, HAS3, as well as actin) scanned on a fluorimager; the upper section is a quantitation of HAS1 mRNA. Values given on the y-axis rep- resent fluorescence units. A, a representative experiment demonstrating that FLS readily respond to IL-1␤ and PMA treatment with the up-regulation of the gene HAS1; at the same time, mRNA levels of HAS2 and HAS3 remain mainly unchanged. More importantly, this figure demonstrates the inhibi- tory effect of leflunomide on IL-1␤- and PMA-induced HAS1 mRNA ac- cumulation. B, Demonstrates that 5 ␮M leflunomide is sufficient to signif- icantly reduce IL-1␤-induced HAS1 mRNA levels. Increasing leflunomide to 50 ␮M (Lef 50 ϩ IL-1␤) results in Ն80% inhibition. C, data demon- strating that IL-1␤ does not affect mRNA levels of hyaluronidases. Shown is a representative experiment in which FLS were left untreated (column Medium), treated with TGF-␤ (1 ng/ml), or treated with IL-1␤ (5 ng/ml) FIGURE 3. Leflunomide inhibits induced HAS1 mRNA levels dose de- for 6 h. As demonsrated in this figure, IL-1␤ treatment does not result in pendently; the genes encoding hyaluronidases are not affected by IL-1␤ changes of the mRNA levels of the genes encoding hyaluronidases. As treatment. FLS were left untreated or stimulated with IL-1␤ (5 ng/ml) or demonstrated here in synoviocytes, mRNA for HYAL1, HYAL2, and PMA (2.5 ng/ml) for 6 h. Where indicated, leflunomide (Lef) was added 30 PH-20 are readily detectable, but are unaffected by the IL-1␤ treatment. In min before stimulation. As indicated by the labeling, levels of HAS1, addition, mRNA levels of actin are presented as a demonstration of equal HAS2, and HAS3 mRNA were determined by RT-PCR and quantitated by loading of mRNA. 7380 LEFLUNOMIDE PREVENTS HAS1 ACTIVATION

IL-1-induced HAS1 activation relies on tyrosine kinase activation Besides being an NF-␬B inhibitor, leflunomide has been shown to interfere with tyrosine kinase activities. To explore the involve- ment of protein tyrosine kinases in IL-1␤-induced HAS1 activa- tion, genistein and the stable erbstatin analog 2,5-dihydroxymeth- ylcinnamate, both of which have been shown to function as tyrosine kinase inhibitors, were used (41, 42). Shown in Fig. 5 are the results of an experiment in which FLS were treated with genistein and erbstatin, followed by stimulation with IL-1␤. De- FIGURE 4. A, IL-1␤ is a potent inducer of NF-␬B translocation in FLS. picted in Fig. 5 are mRNA levels for the genes HAS1 and actin, as ␤ Shown is a representative EMSA experiment demonstrating that IL-1 indicated on the right side of this figure. As demonstrated in this ␬ rapidly leads to release and translocation of NF- B. Although 15 min of figure, both tyrosine kinase inhibitors suppress IL-1␤-induced stimulation with IL-1␤ were sufficient to result in the detection of consid- HAS1 mRNA accumulation. Although no significant effect was erable amounts of NF-␬B translocated to the nucleus, maximal levels of ␮ NF-␬B binding can be observed 30–45 min after addition of IL-1␤. The obtained when genistein was added at concentrations of 5 M, a time FLS were exposed to IL-1␤ (5 ng/ml) is indicated on the top of this higher concentration of 50 ␮M genistein repeatedly blocked the figure. The label Free Pr. indicates the column in which nuclear extract was left out of the reaction mixture. The labels on the left side indicate positions of NF-␬B-specific bands (␬B) and nonspecific protein-DNA interactions Downloaded from (NS), respectively. B, Leflunomide does not inhibit NF-␬B translocation in FLS stimulated with IL-1␤. FLS were left untreated (column MEDIUM), treated with IL-1␤ (5 ng/ml) (column IL-1), or were treated with 5 and 50 ␮M leflunomide, respectively, for 45 min before addition of IL-1␤ (5 ng/ ml) for an additional 45 min (columns LEF 5 ϩ IL-1 and LEF 50 ϩ IL-1, respectively). Equal amounts of nuclear extracts were incubated with 32P- http://www.jimmunol.org/ labeled oligonucleotides resembling the NF-␬B consensus element and separated on a 6% native gel. The comparison of the band intensity of IL-1␤-treated cells with that in cells pretreated with leflunomide indicates that leflunomide at the concentrations used does not prevent IL-1␤-induced NF-␬B activation. C, Demonstration of EMSA specificity. Shown here is a control experiment demonstrating the specificity of NF-␬B-DNA inter- actions in EMSA experiments. Nuclear extract of unstimulated (column MEDIUM) and IL-1␤-stimulated (column IL-1) FLS was separated on a 6% native gel. In competition experiments, aliquots of nuclear extract of

IL-1␤-stimulated FLS were incubated with excess of unlabeled NF-␬B, by guest on September 28, 2021 AP-1, and CRE consensus elements. As shown, only unlabeled NF-␬B oligonucleotides can compete for protein binding. In the column marked Free Pr., no nuclear extract was added to the reaction mixture.

significant reduction in subsequent IL-1␤-induced NF-␬B translocation. Competition experiments as well as supershift experiments were performed, as described earlier (29), ensuring specificity of EMSA experiments. Shown in Fig. 4C are such control experiments, dem- onstrating that while unlabeled NF-␬B/DNA-binding elements can compete for ␬B binding, unlabeled AP-1 or CRE consensus ele- ments cannot.

Reconstituting the pyrimidine synthesis pathway with uridine does not restore IL-1␤-induced HAS1 activation FIGURE 5. IL-1␤-induced HAS1 transcription depends on tyrosine ki- Leflunomide is a known inhibitor of pyrimidine synthesis (39), and nase activation. As indicated on the x-axis, FLS were treated with the two adding exogenous uridine reportedly demonstrated the involve- protein tyrosine kinase inhibitors genistein and erbstatin (50 and 5 ␮M, ment of this mechanism in many leflunomide-mediated effects respectively) for 45 min, followed by stimulation with IL-1␤ (5 ng/ml) for (40). We tested whether such mechanisms might account for the 6 h. An aliquot of the resulting RT-PCR mixture was separated by agarose observed phenomena with regard to the activation of the HAS1 electrophoresis and quantitated on a fluorimager. The positions of mRNA gene. In several independent experiments, the addition of exoge- for HAS1 and actin are indicated on the right side of this figure. Shown is nous uridine did not reverse the leflunomide-mediated effect on one of several experiments demonstrating again that steady state HAS1 IL-1␤-induced HAS1 up-regulation. In some experiments, uridine mRNA levels in unstimulated FLS (column Medium) are low, but are readily up-regulated in cells exposed to IL-1␤ (column IL-1). More im- (100 ␮M) was added together with IL-1␤; in other experiments, at portantly, both tyrosine kinase inhibitors suppress IL-1␤-induced HAS1 the same time as leflunomide. Such experiments (data not shown) mRNA accumulation. Although 50 ␮M genistein suppressed IL-1␤-in- had no marked effect and demonstrated that the suppressive effect duced HAS1 mRNA accumulation by Ն60%, lowering genistein concen- of leflunomide could not be overcome by exogenous uridine, mak- trations to 5 ␮M did not result in significant suppression. Similarly, erb- ing it unlikely that inhibition of pyrimidine synthesis by lefluno- statin at 50 ␮M resulted in Ն90% inhibition, but 5 ␮M erbstatin was also mide accounts for the effect on HAS1. not sufficient to significantly affect IL-1␤-induced HAS1 mRNA levels. The Journal of Immunology 7381

effects of IL-1␤ on HAS1 by Ͼ60%. Similarly, while concentra- Of great importance for the understanding of leflunomide as an tions of 5 ␮M erbstatin were not sufficient to significantly affect anti-inflammatory remedy are also reports demonstrating its inhib- IL-1␤-induced HAS1 mRNA levels, increasing the concentration itory effects on transcription factors that have been shown to be to 50 ␮M resulted in Ͼ90% inhibition. essential for the activation of most proinflammatory genes (36). Arbitrary fluorescence units are given on the y-axis, and culture NF-␬B is a transcription factor that has been studied in great detail. conditions are indicated on the x-axis. In the graphs, HAS1 mRNA The demonstration that leflunomide can act as an inhibitor of levels were normalized using mRNA levels of actin. NF-␬B activation seemed to explain and account for the beneficial effects of leflunomide treatment (22, 38). Interestingly, at least in Discussion T cells, inhibition of NF-␬B activation by leflunomide is also a We tested the effect of leflunomide on noninduced as well as IL- process that can be overcome by addition of exogenous uridine 1␣-, IL-1␤-, and PMA-induced HA activation and release. The (38). Our EMSA data demonstrate clearly that inhibition of IL- presented data demonstrate that in FLS, HAS1 is the gene that is 1␤-induced NF-␬B translocation is not the mode of action of le- readily activated by these stimuli, while the constitutively acti- flunomide in FLS. vated genes encoding HAS2 and HAS3 are not. EMSA experi- IL-1, with its myriad effects on cell signaling, has been shown ments exclude leflunomide-induced inhibition of NF-␬B translo- to activate protein tyrosine kinases (54, 55). Protein tyrosine ki- cation as the mode of action of this drug. Furthermore, inhibition nases are thought to play essential roles in signal transduction in of de novo pyrimidine synthesis by leflunomide as the mode of many cell types and have been demonstrated to play important action could also be ruled out by reconstitution experiments. We roles in cell activation associated with inflammation. Leflunomide

conclude that the effect of leflunomide on HAS1 transcription is has also been described to possess tyrosine kinase inhibitor prop- Downloaded from due to its properties as a tyrosine kinase inhibitor. Such a conclu- erties (56, 57). sion is supported by experiments demonstrating that two tyrosine The concentration of leflunomide necessary for complete HAS1 kinase inhibitors mimic the effects of leflunomide on HAS1 inhibition in in vitro experiments is readily attainable and main- regulation. tained in humans without significant toxicity (58). Furthermore, These and data published earlier (29) demonstrate that HAS1 is the serum concentration of this drug that prevents cardiac allograft

a gene that, in contrast to other HAS genes, is readily activated by rejection has been shown to range from 10 to 100 ␮M (59). As http://www.jimmunol.org/ a series of proinflammatory stimuli. Whether it is also the HAS1 reported for activation of T cells, leflunomide was able to inhibit lck fyn gene product that acts as the ligand for CD44, therefore facilitating Src family (p56 and p59 )-mediated protein kinase at IC50 of ␮ and contributing to undesired cell migration in affected joints, is 125–175 and 22–40 M, respectively, while the IC50 values for currently under investigation. autophosphorylation and phosphorylation of histone 2B were 160 This study has been motivated by our interest in the role that the and 65 ␮M, respectively (56). The same group also demonstrated different HAS genes might play in the pathogenesis of RA. Un- the ability of leflunomide to inhibit protein tyrosine phosphoryla-

doubtedly, HA is essential for many physiological processes; nev- tion induced by anti-CD3 Abs. In such cases, the IC50 values of ertheless, the presence of abnormally large amounts of HA in total intracellular tyrosine phosphorylation ranged from 5 to 45 joints as well as in serum of RA patients is a hallmark of this ␮M. These concentrations are well within the range used in our by guest on September 28, 2021 disorder (15). That HA is much more than an inert matrix molecule experiments and considerably lower than concentrations necessary has become increasingly clear. A series of studies demonstrate, for to inhibit NF-␬B activation (38). example, an exceptional role of HA in the progression of certain The conclusion that the observed effects of leflunomide are in- forms of cancer (43–47). More important in the context of this deed due to inhibition of protein tyrosine kinase activities is sup- manuscript are reports that clearly demonstrate the importance of ported by: 1) our EMSA experiments that exclude the effects of HA in cell adhesion and migration, events that are associated with NF-␬B as the modus operandi; 2) reports that demonstrate the need inflammatory processes also seen in RA. One of the prerequisites for considerably higher concentrations of leflunomide in experi- for molecules involved in inflammation and migration is the ability ments in which this drug has been shown to inhibit the activation to be regulated in a tightly controlled manner. In FLS, only HAS1 of NF-␬B; and 3) the failure to restore IL-1-induced HAS1 acti- seems to fulfill such requirements. Interestingly, plenty of effort vation by exogenous uridine as a measure to reconstitute a possibly was put into understanding the activation and regulation of CD44, blocked pyrimidine synthase pathway. The assumption that IL-1␤- the principal binding partner of HA that has been shown to be induced HAS1 up-regulation is indeed due to the well-described essential for the migration of many cell types (48–50). It is, how- effect of leflunomide on the activation of tyrosine kinase is sup- ever, surprising how little is known about the regulation/activation ported by our data demonstrating that two well-defined protein of HA, the counterpart to CD44. tyrosine kinase inhibitors were similarly effective in suppressing Leflunomide has recently been approved for the treatment of RA IL-1␤-induced HAS1 mRNA accumulation. and has been used for several years with considerable success (51). Collectively, these data suggest that tyrosine kinase activity is It is thought that the molecular mechanisms of leflunomide in RA essential for IL-1␤-induced HAS1 activation. These data also point include interference with IL-2 release and IL-2R binding, modu- at the mechanism by which leflunomide lowers HA production, a lation of Th2-dependent B cell function. Inhibition of various ty- mechanism that might account for the beneficial effects of lefluno- rosine kinases, demonstrated also in animal models, has been re- mide in treating RA. Furthermore, the presented data on the mech- ported to account for effects of leflunomide (52). Other beneficial anism of leflunomide-mediated inhibition of HA up-regulation effects of leflunomide noted by investigators seem to be due to its may be helpful as a tool to further dissect the role of tyrosine inhibition of cell adhesion and cell migration (53). However, the kinases in pathways leading to HAS1 transcription, and might also modus operandi favored by most investigators is the well-docu- contribute to a better understanding of the mode of action of this mented inhibition of de novo pyrimidine synthesis by leflunomide. class of immunomodulatory drug. Data presented in this manuscript seem to exclude such a mech- anism as the mode of action, because replenishing cell culture medium with uridine did not restore IL-1-induced HAS1 Acknowledgments activation. I thank C. Pollaschek for performing RT-PCR experiments. 7382 LEFLUNOMIDE PREVENTS HAS1 ACTIVATION

Disclosures (HAS): hydrocortisone inhibits HAS1 activation by blocking the p38 mitogen- activated protein kinase signalling pathway. Rheumatology 43: 164–169. The author has no financial conflict of interest. 32. Laurent, T. C., U. B. Laurent, and J. R. Fraser. 1995. Functions of hyaluronan. Ann. Rheum. Dis. 54: 429–432. References 33. Laurent, T. C., U. B. Laurent, and J. R. Fraser. 1996. The structure and function of hyaluronan: an overview. Immunol. Cell Biol. 74: A1–A7. 1. Kouri, T. 1985. Etiology of rheumatoid arthritis. Experientia 41: 434–441. 34. Kreil, G. 1995. Hyaluronidases: a group of neglected enzymes. Protein Sci. 4: 2. Vaughan, J. H., T. Kouri, J. Petersen, J. Roudier, and G. H. Rhodes. 1988. On the 1666–1669. etiology of rheumatoid arthritis. Scand. J. Rheumatol. Suppl. 74: 19–28. 35. Flannery, C. R., C. B. Little, C. E. Hughes, and B. Caterson. 1998. Expression 3. Bland, J. H., and C. A. Phillips. 1972. Etiology and pathogenesis of rheumatoid and activity of articular cartilage hyaluronidases. Biochem. Biophys. Res. Com- arthritis and related multisystem diseases. Semin. Arthritis Rheum. 1: 339–359. mun. 251: 824–829. 4. Brahn, E. 1991. Animal models of rheumatoid arthritis: clues to etiology and ␬Binthe treatment. Clin. Orthop. Relat. Res. 265: 42–53. 36. Baeuerle, P. A., and T. Henkel. 1994. Function and activation of NF- immune system. Annu. Rev. Immunol. 12: 141–179. 5. Gerdin, B., and R. Hallgren. 1997. Dynamic role of hyaluronan (HYA) in con- ␬ nective tissue activation and inflammation. J. Intern. Med. 242: 49–55. 37. Schreck, R., K. Albermann, and P. A. Baeuerle. 1992. Nuclear factor B: an 6. Levesque, M. C., and B. F. Haynes. 1999. TNF␣ and IL-4 regulation of hyalu- oxidative stress-responsive transcription factor of eukaryotic cells. Free Radical ronan binding to monocyte CD44 involves posttranslational modification of Res. Commun. 17: 221–237. CD44. Cell. Immunol. 193: 209–218. 38. Manna, S. K., A. Mukhopadhyay, and B. B. Aggarwal. 2000. Leflunomide sup- ␬ 7. Wuthrich, R. P. 1999. The proinflammatory role of hyaluronan-CD44 interactions presses TNF-induced cellular responses: effects on NF- B, activator protein-1, in renal injury. Nephrol. Dial. Transplant. 14: 2554–2556. c-Jun N-terminal protein kinase, and apoptosis. J. Immunol. 165: 5962–5969. 8. Levesque, M. C., and B. F. Haynes. 1997. Cytokine induction of the ability of 39. Cherwinski, H. M., R. G. Cohn, P. Cheung, D. J. Webster, Y. Z. Xu, human monocyte CD44 to bind hyaluronan is mediated primarily by TNF-␣ and J. P. Caulfield, J. M. Young, G. Nakano, and J. T. Ransom. 1995. The immu- is inhibited by IL-4 and IL-13. J. Immunol. 159: 6184–6194. nosuppressant leflunomide inhibits lymphocyte proliferation by inhibiting pyrim- 9. West, D. C., and M. Yaqoob. 1997. Serum hyaluronan levels follow disease idine biosynthesis. J. Pharmacol. Exp. Ther. 275: 1043–1049. activity in vasculitis. Clin. Nephrol. 48: 9–15. 40. Nair, R. V., W. Cao, and R. E. Morris. 1995. Inhibition of smooth muscle cell

10. West, D. C., and S. Kumar. 1989. Hyaluronan and angiogenesis. Ciba Found. proliferation in vitro by leflunomide, a new immunosuppressant, is antagonized Downloaded from Symp. 143: 187–201. by uridine. Immunol. Lett. 48: 77–80. 11. Engstrom-Laurent, A. 1997. Hyaluronan in joint disease. J. Intern. Med. 242: 41. Tetsuka, T., S. K. Srivastava, and A. R. Morrison. 1996. Tyrosine kinase inhib- 57–60. itors, genistein and herbimycin A, do not block interleukin-1␤-induced activation 12. Fraser, J. R., T. C. Laurent, and U. B. Laurent. 1997. Hyaluronan: its nature, of NF-␬B in rat mesangial cells. Biochem. Biophys. Res. Commun. 218: distribution, functions and turnover. J. Intern. Med. 242: 27–33. 808–812. 13. Henderson, E. B., M. Grootveld, A. Farrell, E. C. Smith, P. W. Thompson, and 42. Liu, X. J., L. Yang, Y. Q. Mao, Q. Wang, M. H. Huang, Y. P. Wang, and D. R. Blake. 1991. A pathological role for damaged hyaluronan in synovitis. Ann. H. B. Wu. 2002. Effects of the tyrosine protein kinase inhibitor genistein on the Rheum. Dis. 50: 196–200. proliferation, activation of cultured rat hepatic stellate cells. World http://www.jimmunol.org/ 14. Laurent, T. C., and J. R. Fraser. 1986. The properties and turnover of hyaluronan. J. Gastroenterol. 8: 739–745. Ciba Found. Symp. 124: 9–29. 43. Mytar, B., M. Siedlar, M. Woloszyn, V. Colizzi, and M. Zembala. 2001. Cross- 15. Laurent, T. C., U. B. Laurent, and J. R. Fraser. 1996. Serum hyaluronan as a talk between human monocytes and cancer cells during reactive oxygen inter- disease marker. Ann. Med. 28: 241–253. mediates generation: the essential role of hyaluronan. Int. J. Cancer 94: 727–732. 16. Castor, C. W., R. K. Prince, and M. J. Hazelton. 1966. Hyaluronic acid in human 44. Toole, B. P., T. N. Wight, and M. I. Tammi. 2002. Hyaluronan-cell interactions synovial effusions; a sensitive indicator of altered connective tissue cell function in cancer and vascular disease. J. Biol. Chem. 277: 4593–4596. during inflammation. Arthritis Rheum. 9: 783–794. 45. Liu, N., F. Gao, Z. Han, X. Xu, C. B. Underhill, and L. Zhang. 2001. Hyaluronan 17. Emlen, W., J. Niebur, G. Flanders, and J. Rutledge. 1996. Measurement of serum synthase 3 overexpression promotes the growth of TSU prostate cancer cells. hyaluronic acid in patients with rheumatoid arthritis: correlation with disease Cancer Res. 61: 5207–5214. activity. J. Rheumatol. 23: 974–978. 46. Lipponen, P., S. Aaltomaa, R. Tammi, M. Tammi, U. Agren, and V. M. Kosma. 18. Wang, J. Y., and M. H. Roehrl. 2002. are a potential cause 2001. High stromal hyaluronan level is associated with poor differentiation and

of rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 99: 14362–14367. metastasis in prostate cancer. Eur. J. Cancer 37: 849–856. by guest on September 28, 2021 19. Itano, N., T. Sawai, M. Yoshida, P. Lenas, Y. Yamada, M. Imagawa, 47. Setala, L. P., M. I. Tammi, R. H. Tammi, M. J. Eskelinen, P. K. Lipponen, T. Shinomura, M. Hamaguchi, Y. Yoshida, Y. Ohnuki, et al. 1999. Three iso- U. M. Agren, J. Parkkinen, E. M. Alhava, and V. M. Kosma. 1999. Hyaluronan forms of mammalian hyaluronan synthases have distinct enzymatic properties. expression in gastric cancer cells is associated with local and nodal spread and J. Biol. Chem. 274: 25085–25092. reduced survival rate. Br. J. Cancer 79: 1133–1138. 20. Stuhlmeier, K. M., and C. Pollaschek. 2004. Differential effect of transforming 48. Naor, D., and S. Nedvetzki. 2003. CD44 in rheumatoid arthritis. Arthritis Res. ␤ ␤ growth factor (TGF- ) on the genes encoding hyaluronan synthases and uti- Ther. 5: 105–115. ␤ lization of the p38 MAPK pathway in TGF- -induced hyaluronan synthase 1 49. Goodison, S., V. Urquidi, and D. Tarin. 1999. CD44 cell adhesion molecules. activation. J. Biol. Chem. 279: 8753–8760. Mol. Pathol. 52: 189–196. 21. Smolen, J. S., P. Emery, J. R. Kalden, P. L. Van Riel, M. Dougados, C. V. Strand, 50. Ponta, H., L. Sherman, and P. A. Herrlich. 2003. CD44: from adhesion molecules and F. C. Breedveld. 2004. The efficacy of leflunomide monotherapy in rheuma- to signalling regulators. Nat. Rev. Mol. Cell Biol. 4: 33–45. toid arthritis: towards the goals of disease modifying antirheumatic drug therapy. 51. Breedveld, F. C., and J. M. Dayer. 2000. Leflunomide: mode of action in the J. Rheumatol. Suppl. 71: 13–20. treatment of rheumatoid arthritis. Ann. Rheum. Dis. 59: 841–849. 22. Urushibara, M., H. Takayanagi, T. Koga, S. Kim, M. Isobe, Y. Morishita, Curr. Opin. Investig. Drugs T. Nakagawa, M. Loeffler, T. Kodama, H. Kurosawa, and T. Taniguchi. 2004. 52. Kaplan, M. J. 2001. Leflunomide Aventis Pharma. 2: The antirheumatic drug leflunomide inhibits osteoclastogenesis by interfering 222–230. with receptor activator of NF-␬B ligand-stimulated induction of nuclear factor of 53. Kraan, M. C., B. M. de Koster, J. G. Elferink, W. J. Post, F. C. Breedveld, and activated T cells c1. Arthritis Rheum. 50: 794–804. P. P. Tak. 2000. Inhibition of neutrophil migration soon after initiation of treat- 23. Jakez-Ocampo, J., Y. Richaud-Patin, J. Granados, J. Sanchez-Guerrero, and ment with leflunomide or methotrexate in patients with rheumatoid arthritis: find- L. Llorente. 2004. Weekly leflunomide as monotherapy for recent-onset rheuma- ings in a prospective, randomized, double-blind clinical trial in fifteen patients. toid arthritis. Arthritis Rheum. 51: 147–148. Arthritis Rheum. 43: 1488–1495. 24. Bartlett, R. R., S. Brendel, T. Zielinski, and H. U. Schorlemmer. 1996. Lefluno- 54. Carlson, R. O., and S. H. Aschmies. 1995. Tyrosine kinase activity is essential for ␤ mide, an immunorestoring drug for the therapy of autoimmune disorders, espe- interleukin-1 -stimulated production of interleukin-6 in U373 human astrocy- cially rheumatoid arthritis. Transplant. Proc. 28: 3074–3078. toma cells. J. Neurochem. 65: 2491–2499. 25. Williams, J. W., D. Mital, A. Chong, A. Kottayil, M. Millis, J. Longstreth, 55. Ito, A., H. Shimokawa, T. Kadokami, Y. Fukumoto, M. K. Owada, T. Shiraishi, W. Huang, L. Brady, and S. Jensik. 2002. Experiences with leflunomide in solid R. Nakaike, T. Takayanagi, K. Egashira, and A. Takeshita. 1995. Tyrosine kinase organ transplantation. Transplantation 73: 358–366. inhibitor suppresses coronary arteriosclerotic changes and vasospastic responses ␤ 26. Gummert, J. F., T. Ikonen, and R. E. Morris. 1999. Newer immunosuppressive induced by chronic treatment with interleukin-1 in pigs in vivo. J. Clin. Invest. drugs: a review. J. Am. Soc. Nephrol. 10: 1366–1380. 96: 1288–1294. 27. Yuh, D. D., K. L. Gandy, R. E. Morris, G. Hoyt, J. Gutierrez, B. A. Reitz, and 56. Xu, X., J. W. Williams, E. G. Bremer, A. Finnegan, and A. S. Chong. 1995. R. C. Robbins. 1995. Leflunomide prolongs pulmonary allograft and xenograft Inhibition of protein tyrosine phosphorylation in T cells by a novel immunosup- survival. J. Heart Lung Transplant. 14: 1136–1144. pressive agent, leflunomide. J. Biol. Chem. 270: 12398–12403. 28. Partsch, G., and M. Matucci-Cerinic. 1990. Effect of capsaicin on the release of 57. Siemasko, K., A. S. Chong, H. M. Jack, H. Gong, J. W. Williams, and substance P from rheumatoid arthritis and osteoarthritis synoviocytes in vitro. A. Finnegan. 1998. Inhibition of JAK3 and STAT6 tyrosine phosphorylation by Ann. Rheum. Dis. 49: 653. the immunosuppressive drug leflunomide leads to a block in IgG1 production. 29. Stuhlmeier, K. M., C. Tarn, V. Csizmadia, and F. H. Bach. 1996. Selective sup- J. Immunol. 160: 1581–1588. pression of endothelial cell activation by arachidonic acid. Eur. J. Immunol. 26: 58. Hsi, E. D., J. N. Siegel, Y. Minami, E. T. Luong, R. D. Klausner, and 1417–1423. L. E. Samelson. 1989. T cell activation induces rapid tyrosine phosphorylation of 30. Spicer, A. P. 2001. In vitro assays for hyaluronan synthase. Methods Mol. Biol. a limited number of cellular substrates. J. Biol. Chem. 264: 10836–10842. 171: 373–382. 59. Tsygankov, A. Y., B. M. Broker, J. Fargnoli, J. A. Ledbetter, and J. B. Bolen. 31. Stuhlmeier, K. M., and C. Pollaschek. 2004. Glucocorticoids inhibit induced and 1992. Activation of tyrosine kinase p60fyn following T cell antigen receptor cross- non-induced mRNA accumulation of genes encoding hyaluronan synthases linking. J. Biol. Chem. 267: 18259–18262.