Sung-Ho Ryu,4 Yeun-Jun Chung,1 Chul-Soo Cho,1 and Wan-Uk Kim1
Objective. To investigate whether sulforaphane(SFN), an isothiocyanate derived from cruciferous veg-etables such as broccoli, regulates synoviocyte hyperpla-sia and T cell activation in rheumatoid arthritis (RA).
Methods. Synoviocyte survival was assessed byMTT assay. The levels of Bcl-2, Bax, p53, and pAkt weredetermined by Western blot analysis. Cytokine concen-trations in culture supernatants from mononuclear cellswere analyzed by enzyme-linked immunosorbent assay.The in vivo effects of SFN were examined in mice withexperimentally induced arthritis.
Results. SFN induced synoviocyte apoptosis bymodulating the expression of Bcl-2/Bax, p53, and pAkt.In addition, nonapoptotic doses of SFN inhibited T cellproliferation and the production of interleukin-17 (IL-17) and tumor necrosis factor � (TNF�) by RA CD4� Tcells stimulated with anti-CD3 antibody. Anti-CD3antibody–induced increases in the expression of retinoicacid–related orphan receptor �t and T-bet were alsorepressed by SFN. Moreover, the intraperitoneal admin-istration of SFN to mice suppressed the clinical severityof arthritis induced by injection of type II collagen (CII),
the anti-CII antibody levels, and the T cell responses toCII. The production of IL-17, TNF�, IL-6, andinterferon-� by lymph node cells and spleen cells fromthese mice was markedly reduced by treatment withSFN. Anti-CII antibody–induced arthritis in mice wasalso alleviated by SFN injection.
Conclusion. SFN was found to inhibit synovialhyperplasia, activated T cell proliferation, and the pro-duction of IL-17 and TNF� by rheumatoid T cells invitro. The antiarthritic and immune regulatory effects ofSFN, which were confirmed in vivo, suggest that SFNmay offer a possible treatment option for RA.
A number of xenobiotics, which are foreign toliving organisms, can be modified by phase I enzymessuch as cytochrome P450 oxygenase to become ex-tremely toxic products in the human body (1). Theso-called phase II enzymes, such as glucuronosyltrans-ferase and NADPH quinone oxidoreductase 1, catalyzethe further biotransformations of xenobiotics (1), lead-ing to their conjugation, methylation, acetylation, andconversion to less toxic, more soluble, and inactivemetabolites that are readily excreted (1). These enzymesalso mediate a variety of cytoprotective reactions againstelectrophiles and reactive oxygen species (2). In thisregard, the pharmacologic inducers of phase II enzymeactivity offer a new means of preventing oxidative dam-age (2,3).
Sulforaphane (SFN), a member of the isothiocya-nate family, is a highly potent inducer of phase IIenzymes (3). It is abundantly present in cruciferousvegetables, such as broccoli, cauliflower, and radishes(3). Over a decade ago, SFN was identified as a chemo-preventive agent (4–12). Epidemiologic studies havesuggested that people who consume cruciferous vegeta-bles have a lower risk of developing cancer (4–6). SFN is
Supported by grants from the National Research Foundationof Korea (2009-0080087 and KRF-2008-220-E00025).
1Jin-Sun Kong, MS, Seung-Ah Yoo, MS, Yeun-Jun Chung,MD, PhD, Chul-Soo Cho, MD, PhD, Wan-Uk Kim, MD, PhD:Catholic University of Korea, Seoul, South Korea; 2Hyun-Sook Kim,MD, PhD: Chosun University of Korea, Gwangju, South Korea; 3HyunAh Kim, MD, PhD: Hallym University Sacred Heart Hospital, An-yang, Kyunggi-do, South Korea; 4Kyungmoo Yea, PhD, Sung-Ho Ryu,PhD: Pohang University of Science and Technology, Pohang, SouthKorea.
Ms Kong and Ms Yoo contributed equally to this work.Address correspondence and reprint requests to Wan-Uk
Kim, MD, PhD, Division of Rheumatology, Department of InternalMedicine, Catholic University of Korea, St. Vincent’s Hospital, 93Chi-Dong, Suwon 442-723, South Korea. E-mail: [email protected].
Submitted for publication July 1, 2008; accepted in revisedform August 31, 2009.
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known to affect various stages of cancer development,and it has been reported to prevent, delay, or evenreverse carcinogenesis in vitro and in vivo (3–7). Theseeffects of SFN are primarily attributed to its modulationof the activities of phase II enzymes (7), which convertcarcinogens to inactive metabolites and prevent themfrom interacting with DNA. SFN also traps free radicalsand induces cell cycle arrest (8,9), which constituteadditional anticancer mechanisms. Moreover, SFN in-duces apoptosis of cancer cells by regulating multipletargets (10–12). For example, SFN can activateapoptosis-inducing genes, including those for caspases(10), p53 (11), and the nuclear factor signaling pathway(12). A decrease in Bcl-2 expression and an increase inBax expression have also been demonstrated duringSFN-induced apoptosis (10–12).
Rheumatoid arthritis (RA) is characterized by atumor-like expansion of the synovium that is composedof proliferating synoviocytes and infiltrating leukocytes,including T cells and B cells, which are likely activated byautoantigens (13). Interleukin-17 (IL-17)–producing Tcells (the so-called Th17 cells) have emerged as one ofthe immune cells that are associated with the initiationand perpetuation of RA (14), and the modulation ofIL-17 has been demonstrated to be effective in thesuppression of arthritis (15,16). Resident synoviocytesalso participate in the chronic inflammatory responsesof RA, and in fact, these cells represent the major cellpopulation in invasive pannus (13,17). RA synoviocyteshave the potential to produce matrix-degrading enzymesand several cytokines, including IL-6 and IL-8 (13,17).Moreover, fibroblast-like synoviocytes (FLS) from RApatients proliferate abnormally, invade the local envi-ronment, and exhibit many of the characteristics oftumor cells (18,19). For this reason, inducers of apopto-sis that prevent synoviocyte proliferation, such as anti-Fas antibody, have been investigated for their ability toretard synovial growth and joint destruction in mice (20).
Given the pathologic similarities between RAsynoviocytes and tumor cells, we considered it relevantto test the effects of SFN on synoviocyte apoptosis. Inthis study, we first demonstrated that SFN inhibitedsynoviocyte survival and T cell proliferation. The pro-duction of IL-17 and tumor necrosis factor � (TNF�) byRA T cells was also repressed by SFN. In addition, theadministration of SFN to mice with experimental arthri-tis increased synoviocyte apoptosis, while it decreasedchronic synovitis, autoantibody formation, and antigen-specific T cell proliferation. The production of IL-17,TNF�, IL-6, and interferon-� (IFN�) by lymph nodecells and spleen cells was also reduced in SFN-treated
mice. These findings suggest that SFN might be usefulfor the treatment of RA in humans.
MATERIALS AND METHODS
Isolation and culture of mononuclear cells and FLSfrom RA patients. Heparinized peripheral blood (100 ml) wascollected under aseptic conditions from patients with RA.Informed consent for use of the cells was obtained from all RApatients included in this study. This study was approved by theinstitutional review committees of the Catholic Medical Center(Seoul, South Korea).
Peripheral blood mononuclear cells (PBMCs) wereisolated by density-gradient centrifugation on Ficoll-Hypaque.PBMCs or CD4� cells that were sorted with the use ofanti-CD4 microbeads (Miltenyi Biotec, Auburn, CA) werethen cultured in RPMI 1640 medium supplemented with 10%fetal bovine serum (FBS; Gibco BRL, Grand Island, NY), 100units/ml of penicillin, 100 �g/ml of streptomycin, and 2 mML-glutamine. Each culture was performed in triplicate in 96-well plates.
Cells were incubated at 37°C for the indicated times inan atmosphere containing 5% CO2 with various concentrationsof SFN (LKT Laboratories, St. Paul, MN), which ranged from0.01 �M to 100 �M. In some experiments, the cells werestimulated with phytohemagglutinin (PHA; Sigma, St. Louis,MO), lipopolysaccharide (LPS; Sigma), or anti-CD3 antibodyplus anti-CD28 antibody (both from BD Biosciences, SanDiego, CA) in the absence or presence of SFN. After incuba-tion for 24 hours, the cell-free supernatants were collected andstored at –20°C until required for assay.
FLS were prepared from the synovial tissues of RApatients, as described previously (21). Cells obtained frompassages 3–8 were seeded into 24-well plates at a density of 2 �104 cells/well in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 5% FBS, 100 units/ml of peni-cillin, 100 �g/ml of streptomycin, and 2 mM L-glutamine, andthen they were incubated at 37°C.
MTT assay for cell viability. FLS were seeded in24-well plates at a density of 2 � 104 cells/well in DMEMsupplemented with 5% FBS in the absence or presence of SFN(5–100 �M). After 24 or 48 hours of incubation, 2.5 mg/ml ofMTT solution (Sigma) was added to each of the wells, and thecells were then incubated for 2 hours. The reaction wasstopped by removing the MTT solution. The absorbance wasread at 540 nm with a microplate reader (Molecular Devices,Palo Alto, CA).
Detection of apoptotic events. To quantify the apopto-sis of cultured FLS, APOPercentage apoptosis assay kits(Biocolor, Newtownabbey, UK) were used according to themanufacturer’s instructions. This assay uses a dye that stainscells as they undergo membrane “flip-flop” events when phos-phatidylserine is translocated to the outer leaflet. Digitalimages of the APOPercentage dye–labeled cells, which appearbright pink against a white background, were used to quantifythe number of apoptotic FLS. In addition, cell death wasassessed by flow cytometry using propidium iodide. Briefly,FLS (5 � 105) were centrifuged at 200g for 10 minutes,resuspended in 500 �l of hypotonic fluorochrome solution
160 KONG ET AL
containing 50 �g/ml of propidium iodide, and then incubatedon ice for 15–30 minutes before analysis. A minimum of 10,000events were collected and analyzed on a FACScan cytometer(Becton Dickinson, San Jose, CA).
Apoptotic cells in the joints of arthritic mice were alsodetected by the TUNEL method, which relies on DNA frag-mentation. Briefly, joint tissues were fixed, paraffin-embedded,sectioned, and stained by using an In Situ Cell Death Detec-tion kit (Roche Applied Science, Indianapolis, IN) accordingto the manufacturer’s protocol. Sections were counterstainedwith hematoxylin, and TUNEL� cells were counted under alight microscope at 400� magnification. The ratio of the totalnumber of positive cells to the total cells counted was deter-mined.
Western blot analyses of Bcl-2, Bax, p53, and pAkt.RA FLS were incubated for 24 hours in 5% FCS–DMEM, andvarious concentrations of SFN (0–100 �M) were added to thecells for the indicated times. FLS were then washed twice inphosphate buffered saline and dissolved in sample buffer. Theproteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocel-lulose membranes. After immunoblot analysis with anti–Bcl-2antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bax antibody (Santa Cruz Biotechnology), anti-p53 antibody(Santa Cruz Biotechnology), or anti-pAkt antibody (Ser473;Cell Signaling Laboratories, Beverly, MA), the membraneswere stripped and incubated with anti–�-actin antibody inorder to quantify total proteins.
Cytokine enzyme-linked immunosorbent assay(ELISA). The amounts of IL-17, TNF�, IL-6, IFN�, and IL-10released into the supernatants of the cultured cells weremeasured by ELISA, as previously described (21).
RNA isolation and real-time polymerase chain reac-tion (PCR) analysis. Total RNA was extracted from CD4� Tcells with the use of RNAzol B (Biotex Laboratories, SanAntonio, TX) and then reverse-transcribed into complemen-tary DNA (cDNA) with RevertAid Moloney murine leukemiavirus reverse transcriptase (Fermentas, Hanover, MD), ran-dom hexamer (Takara, Shiga, Japan), and 2 �g of total RNA.The cDNA was adjusted to contain equal numbers of �-actintranscripts, as determined by real-time PCR.
The following primer sequences were used: for IL-17,5�-TGGAGGCCATAGTGAAGG-3� and 5�-GGCCACATG-GTGGACAAT-3�; for TNF�, 5�-CTGTAGCCCATGTTGT-AGCAAAC-3� and 5�-GACCTGGGAGTAGATGAGGTAC-AG-3�; for retinoic acid–related orphan receptor �t (ROR�t),5�-TTTTCCGAGGATGAGATTGC-3� and 5�-CTTTCCAC-ATGCTGGCTACA-3�; for T-bet, 5�-AGAAAGTCTTGGG-CATGAAG-3� and 5�-TCAGGGATTAACACACATGC-3�;for GATA-3, 5�-AGGAGCAGTATCATGAAGCC-3� and5�-TCAGTGGTTGGAACACAGAC-3�; and for �-actin, 5�-GAATCTCCGACCACCACTAC-3� and 5�-AAGGTGCTC-AGGTCATTCTC-3�. The transcripts for each gene werequantified using an Mx3000 PCR instrument (Stratagene,Cedar Creek, TX). The cDNA (2 �l) was mixed with a totalvolume of 20 �l of SYBR Green Master Mix (Stratagene). ThePCR amplification protocol involved 40 cycles of denaturationat 95°C for 30 seconds, primer annealing at 55°C or 60°C for 30seconds, and extension at 72°C for 30 seconds. Nonspecificamplification was detected by analysis of the melting curve.
Assessment of T cell proliferation. PBMCs from theRA patients were stimulated with PHA (5 �g/ml) or withanti-CD3 antibody (1 �g/ml) plus anti-CD28 antibody (1�g/ml), and then cultured in triplicate for 24 or 48 hours on96-well plates at a density of 5 � 105 cells/well. To assess T cellproliferation, 1 �Ci/well of 3H-thymidine (NEN, Boston, MA)was added to each well. Sixteen hours later, the cells wereharvested onto glass-fiber filters and counted using a Matrix 96direct ionization beta counter, as described previously (22).Data are presented as the total counts per minute.
Determination of the effect of SFN on collagen-induced arthritis (CIA). Male DBA/1 mice (The JacksonLaboratory, Bar Harbor, ME) were immunized with bovinetype II collagen (CII; Chondrex, Redmond, WA) at 7–8 weeksof age, as described previously (23). Three weeks after theprimary immunization, mice with CIA were injected withvarious concentrations of SFN (300 �M, 1.5 mM, and 7.5 mM)every other day for 5 weeks. For this procedure, 200 �l of SFNat concentrations of 12.8, 63.8, and 318.8 mg/ml/kg was admin-istered intraperitoneally. Control mice received vehicle alone.
Arthritis severity was determined by visual inspection,as described previously (23). In some experiments, mice werekilled on day 42 for histologic analysis, as described previously(23). Paws and ankles, with the exception of the hind feet thatreceived a booster immunization, were harvested. The degreeof synovial hyperplasia and inflammation in the joints wasdetermined using a standard scoring protocol, in which theseverity was scored on a scale of 0–3, where 0 � absent, 1 �weak, 2 � moderate, and 3 � severe. The maximum possiblescore per mouse was 9.
Immunohistochemical analysis for von Willebrandfactor (vWF). Immunostaining of mouse ankle joints wasperformed using the 5-�m sections obtained from theformalin-fixed, paraffin-embedded blocks. Nonspecific bindingwas blocked by treating the sections with 10% normal goatserum at 37°C for 60 minutes. The sections were then incu-bated overnight at 4°C with rabbit anti-vWF antibody (Abcam,Cambridge, MA) and then for 60 minutes at room temperaturewith biotinylated secondary antibody (Santa Cruz Biotechnol-ogy). Sections were then treated for 30 minutes at roomtemperature with peroxidase-conjugated streptavidin (Dako,Carpinteria, CA), and with 3,3�-diaminobenzidine to reveal theantigen. Hematoxylin was used as counterstain.
Liquid chromatography mass spectrometry (LC-MS).Plasma SFN concentrations were determined as previouslydescribed (24). Plasma samples were passed through Centriconfilters (Millipore, Billerica, MA) at a temperature of 4°C toremove molecules with molecular masses �3 kd. All massanalyses were performed with a nano–LC-MS system thatconsisted of an Ultimate high-performance liquid chromatog-raphy (HPLC) system (LC Packings, Amsterdam, The Neth-erlands) and a Q-Trap 2000 hybrid tandem mass spectrometer(Applied Biosystems/MDS Sciex; Concord, Ontario, Canada)equipped with a nano–electrospray ionization (nano-ESI)source. A nanoscale reverse-phase chromatography analyticalcolumn (LC Packings) was used for high-resolution separation.Mobile phase A consisted of HPLC-grade water with 0.1%formic acid, and mobile phase B consisted of 100% HPLC-grade acetonitrile with 0.1% formic acid. Separation wasperformed at a flow rate of 500 nl/minute. The ion sprayneedle was maintained at 1.8 kV under the positive mode. The
SFN REGULATION OF RHEUMATOID INFLAMMATION 161
analyte was detected by monitoring the mass/charge (m/z)178-to-72 mass transition in the multiple reaction–monitoringscan mode.
Assay for autoimmune response to CII in mice withCIA. Sera from mice with CIA were collected 42 days after theprimary immunization and were stored at –20°C until assayed.The levels of IgG antibody to CII in the sera were determinedusing a commercially available ELISA kit (Chondrex). T cellproliferative responses were also assessed in lymph node cellsand spleen cells obtained 6 weeks after the primary immuni-zation, as previously described (23). Rates of T cell prolifera-tion were determined using the method described above forthe RA PBMC study. In addition, concentrations of IL-17,TNF�, IL-6, IFN�, and IL-10 in culture supernatants of lymphnode cells and spleen cells that had been stimulated with CIIfor 48 hours were measured by ELISA.
Induction of collagen antibody–induced arthritis(CAIA). Eight-week-old male BALB/c mice were used for theinduction of CAIA. Anticollagen antibodies (2 mg; Chondrex)were injected intravenously into 2 groups of mice, and 3 dayslater, 50 �g of LPS was administered intraperitoneally. Micewere monitored and evaluated daily for arthritis. The clinicalseverity of arthritis was graded in each limb using a scale of0–4, as described previously (25), with a maximum score of 16for each animal. During the course of the arthritis, swelling ofthe front and hind feet was also measured every day usingmicrocalipers. The increase in diameter of the arthritic ankle atspecific time points over that on day 0 was defined as the pawthickness index, and this value is presented as the percentage.
Statistical analysis. Data are expressed as the mean �SD. Comparisons of the numerical data between groups wereperformed by Mann-Whitney paired or unpaired U test. Pvalues less than 0.05 were considered statistically significant.
RESULTS
SFN induction of synoviocyte apoptosis by regu-lation of the expression of Bcl-2 and Bax. To study theeffect of SFN on apoptotic death, RA FLS were culturedwith various concentrations of SFN. As shown in Figure1A, SFN decreased synoviocyte survival in a dose-dependent and time-dependent manner. The maximumdecrease observed was to 51% of the basal viability for100 �M SFN. SFN-induced cell death was attributable toapoptosis because the membrane “flip-flop” events wereremarkably induced by 50 �M SFN (Figure 1B). Celldeath was confirmed by flow cytometry using propidiumiodide (Figure 1C).
The Bcl-2 family members are critical to theregulation of survival via the modulation of mitochon-drial integrity (26). Moreover, enhanced expression ofthe antiapoptotic Bcl-2 family members, but not theproapoptotic members, including Bad and Bax, havebeen implicated in the pathogenesis of RA (26). Asshown in Figures 2A, B, and D, expression of antiapop-totic Bcl-2 in RA FLS was reduced in a dose- andtime-dependent manner by treatment with SFN. Incontrast, the expression of proapoptotic p53 and Bax wasincreased by SFN in a time- and dose-dependent manner(Figures 2A, B, and D), indicating that SFN may induceFLS death by regulating the expression of Bcl-2, p53,and Bax.
Figure 1. Sulforaphane (SFN) induction of synoviocyte apoptosis. A, Decreased synoviocytesurvival following treatment with SFN. Fibroblast-like synoviocytes (FLS) from rheumatoidarthritis (RA) patients were treated for 24 or 48 hours with increasing concentrations of SFN(5–100 �M). Cell viability was determined by MTT assay. Values are the mean and SD of 6independent experiments. � � P � 0.05 versus untreated cells. B and C, Induction of synoviocyteapoptosis following treatment with SFN. RA FLS (5 � 104 cells) were cultured for 24 hours with1% fetal calf serum/Dulbecco’s modified Eagle’s medium in the presence of 50 �M SFN. Cellapoptosis was determined by APOPercentage apoptosis assay (a colorimetric method) (B) and flowcytometry using propidium iodide (PI) (C). Under phase-contrast microscopy (B), FLS treated withSFN 50 �M became spherical, shrunken, and detached from the bottoms of the culture plates,whereas untreated cells retained a bipolar appearance. Bright pink cells in the bottom right imageare apoptotic cells.
162 KONG ET AL
The activation of pAkt maintains mitochondrialintegrity via the up-regulation of Bcl-2 expression (27),but it decreases the cellular levels and transcriptionalactivity of p53 (28,29). Since Akt is critical for thesurvival of RA FLS (26), we attempted to determinewhether SFN regulates the phosphorylation of Akt inRA FLS. Our findings indicated that the pAkt activitywas dose-dependently decreased by SFN (Figures 2Cand D). These data, together with the data from previ-ous reports (26–29), suggest that SFN induces a de-crease in pAkt activity in RA FLS, which in turn, altersthe expression of Bcl-2, p53, and Bax, leading to syno-viocyte death.
Suppression of proinflammatory cytokine pro-duction and T cell proliferation by SFN. Activated Tlymphocytes that are possibly triggered by autoantigensalso contribute to joint destruction in RA (13–15,30). Asshown in Figure 3A, SFN (1–100 �M) dose-dependentlyincreased the death of unstimulated RA T cells after 48hours of incubation. However, in contrast to FLS, treat-ment of PBMCs (data not shown) and sorted RA T cells(Figure 3A) with �50 �M SFN for 24 hours did notaffect cell viability. Thus, we next investigated whether anonapoptotic concentration of SFN modulates the acti-vation and proliferation of RA T cells.
As shown in Figure 3B, when the RA PBMCswere stimulated with PHA or with anti-CD3 antibodyplus anti-CD28 antibody for 24 hours, SFN treatment(0.01–10 �M) markedly decreased the amount of IL-17(Figure 3B) and TNF� (data not shown) secreted by
these cells, whereas it did not suppress the levels ofIL-10 (data not shown). In addition, SFN nearly com-pletely inhibited the production of IL-17 and TNF� byfreshly sorted CD4� cells stimulated with anti-CD3antibody plus anti-CD28 antibody (Figure 3C).
The levels of expression of messenger RNA(mRNA) for IL-17 and TNF� in RA CD4� T cellsactivated by CD3 ligation also were dose-dependentlydecreased by SFN treatment (Figure 3D). Similar toT-bet in Th1 cells and GATA-3 in Th2 cells, ROR�t,which is encoded by the RORC gene, was recentlyshown to regulate Th17 cell lineage differentiation (31).As shown in Figure 3E, SFN dose-dependently inhibitedthe anti-CD3 antibody–induced increases in ROR�t andT-bet mRNA expression in RA T cells but did not affectGATA-3 mRNA expression, suggesting that SFN ham-pers the antigen-triggered production of IL-17 throughthe transcriptional regulation of ROR�t.
In contrast, SFN (0.1–10 �M) dose-dependentlyrepressed to basal levels the proliferative response ofRA PBMCs that had been stimulated with PHA or withanti-CD3 antibody plus anti-CD28 antibody (Figure 3F).A nontoxic dose of SFN (0.1–30 �M) also modestlyarrested cell-cycle progression in the G1 phase in bothnormal and RA T cells (data not shown), as determinedby flow cytometry. Taken together, the findings showthat SFN inhibited the activation and proliferation ofRA T cells that secrete such proinflammatory cytokinesas IL-17 and TNF�.
Figure 2. Sulforaphane (SFN) regulation of Bcl-2, Bax, p53, and pAkt expression in rheumatoidarthritis (RA) synoviocytes. A, Changes in the expression of Bcl-2, p53, and Bax in RAfibroblast-like synoviocytes (FLS) treated for 12 hours with 5–100 �M SFN, as determined byWestern blotting. B, Time-dependent response of the expression of Bcl-2, p53, and Bax in RA FLStreated with 100 �M SFN for the indicated times. C, Decreased phosphorylation of Akt in RA FLStreated for 10 minutes with 10–100 �M SFN. Western blots were probed with anti-pAkt (Ser473).D, The optical density (OD) ratio of the expression of Bcl-2, Bax, or pAkt to �-actin. Values arethe mean and SD of 4 separate experiments. � � P � 0.05 versus untreated cells.
SFN REGULATION OF RHEUMATOID INFLAMMATION 163
SFN inhibition of CIA in mice. We next exam-ined whether SFN ameliorates the severity of experi-mental arthritis in vivo. As shown in Figure 4A, an
intraperitoneal injection of SFN (300 �M, 1.5 mM, and7.5 mM) every other day for 5 weeks inhibited thearthritis severity in a dose-dependent manner.
On histologic examination of the joints, we alsofound that the paws and ankles of mice treated with SFN(1.5 mM) exhibited lower degrees of inflammation,synovial hyperplasia, pannus formation, and bone de-struction than did mice treated with vehicle only (Fig-ures 4B and C). Joints from a mouse treated with vehiclealone show marked erosive (arrows in Figure 4B, part b)and destructive arthritis. Hematoxylin and eosin staining(Figure 4B, parts a, b, and d) revealed that the articularspaces were filled with inflammatory cells and spindle-shaped FLS (arrowheads in Figure 4B, part b). Exposureof the subchondral bone as well as loss of calcifiedcartilage in the ankle joints were also observed (arrowsin Figure 4B, part d). In addition, von Willebrand factorstaining of the arthritic synovium (Figure 4B, part c)revealed neovascularization with pannus formation (ar-rows in Figure 4B, part c; positive cells indicated inbrown). In contrast, the joints of a mouse treated withSFN (1.5 mM) (Figure 4B, parts e and f) demonstratesthat the synovium is normal (arrows in Figure 4B, part e)and that the articular cartilage in the ankle joints is wellpreserved (arrows in Figure 4B, part f).
On TUNEL staining (Figure 4D), the number ofpositive synoviocytes was much higher in mice treatedwith 7.5 mM SFN (n � 7) than in control mice (n � 7)(mean � SD 24.7 � 8.8% versus 3.3 � 2.9%; P � 0.001),suggesting that SFN inhibits CIA, at least in part, byinducing synoviocyte apoptosis. No apparent adverseeffects, including weight loss, alterations in physicalappearance, or changes in behavior, were noted in themice treated with 7.5 mM SFN during the course ofthese experiments (data not shown). These results indi-cate that the administration of SFN suppresses theclinical and pathologic severity of arthritis in micewithout inducing serious side effects.
To determine the pharmacokinetics of SFN,plasma levels of SFN in mice were assessed by LC-MSafter a single intraperitoneal injection of 7.5 mM SFN.The average levels of SFN up to 12 hours after injectionare illustrated in Figure 4E. The mean plasma concen-tration was increased to 210 �M at 1 hour, and then itrapidly decreased to 24 �M at 4 hours after dosing.These findings are consistent with those in a previousstudy (24). The data indicate that intraperitoneal injec-tion of 7.5 mM SFN results in an increase in its plasmaconcentration up to the effective level in vitro.
Figure 3. Sulforaphane (SFN) inhibition of the activation and prolif-eration of rheumatoid arthritis (RA) T cells. A, Viability of T cellstreated with SFN. CD4� cells from RA patients (n � 5) were treatedwith SFN for 24 or 48 hours and then subjected to MTT assay. B andC, SFN inhibition of interleukin-17 (IL-17) and tumor necrosis factor� (TNF�) production. Peripheral blood mononuclear cells (1 �106/well) (B) or CD4� T cells (5 � 105/well) (C) from RA patients(n � 4) were stimulated with phytohemagglutinin (PHA; 5 �g/ml) orwith anti-CD3 antibody (1 �g/ml) plus anti-CD28 antibody (1 �g/ml)for 24 hours in the presence of SFN. Levels of IL-17 and TNF� inculture supernatants were determined by enzyme-linked immunosor-bent assay. � � P � 0.05 versus stimulated cells without SFN. D, SFNabrogation of the increased expression of mRNA for IL-17 and TNF�(ratio of cytokine to �-actin) in CD4� T cells from RA patients (n �4) stimulated with anti-CD3 plus anti-CD28 antibodies. � � P � 0.05versus antibody-stimulated cells in the absence of SFN. E, Real-timepolymerase chain reaction analysis of the expression of mRNA forretinoic acid–related orphan receptor �t (ROR�t), T-bet, andGATA-3, as measured in freshly sorted CD4� T cells from RApatients (n � 4) stimulated with anti-CD3 plus anti-CD28 antibodies inthe absence or presence of SFN. � � P � 0.05 versus antibody-stimulated cells without SFN. F, Suppression of RA T cell proliferationby SFN. Proliferative responses of T cells from RA patients (n � 4)stimulated with anti-CD3 plus anti-CD28 antibodies, PHA, or nostimuli in the presence of increasing concentrations of SFN weredetermined by the incorporation of 3H-thymidine (cpm). Values arethe mean � SD.
164 KONG ET AL
Effects of SFN on the autoimmune and cytokineresponses in mice with CIA. Based on the antiarthriticeffect of SFN, we next investigated changes in the levelsof pro- and antiinflammatory cytokines, as well as theCII-specific autoimmune responses, in mice treated withSFN. We found that the levels of IgG antibody to CIIwere diminished in sera from mice treated with SFN ascompared with control mice (Figure 5A). The T cellproliferative response to CII in lymph node cells and
spleen cells was also significantly lower in mice admin-istered SFN (1.5 mM) than in control mice (Figure 5B),although the spontaneous proliferation of T cells in theabsence of CII was not different between the 2 groups(data not shown).
In parallel studies, lymph node cells and spleencells from mice treated with SFN (1.5 mM) were foundto secrete less IL-17, TNF�, IL-6, and IFN�, as com-pared with those from untreated control mice, after
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c e
d f
Figure 4. Suppression of collagen-induced arthritis (CIA) by sulforaphane (SFN). A, Mean arthritis index in mice with CIA treated with variousconcentrations of SFN or vehicle (phosphate buffered saline). SFN or vehicle was administered intraperitoneally to mice with CIA on alternate daysfor 5 weeks. The mean arthritis index was calculated at the indicated time points. � � P � 0.01 versus vehicle-treated controls. B, Histopathologicfeatures of the joints of mice treated with vehicle (a–d) or 1.5 mM SFN (e and f). Images in b and c are higher-magnification views of the red andblack boxed areas in a, respectively. Arrows in b–f and arrowheads in b are described in detail in the Results. (Hematoxylin and eosin stained; originalmagnification � 40 in a and e; � 100 in b, d, and f; � 400 in c.) C, Mean histology score for the degree of inflammation (IFM), synovial hyperplasia(SH), and pannus formation (PF) in mice treated with vehicle or with 1.5 mM SFN. Values are the mean and SD of 6 mice per group. � � P � 0.001versus vehicle-treated controls. D, Determination of synoviocyte apoptosis by the TUNEL method. Positive staining of nuclei (brown) is shown injoint sections from an SFN-treated and a vehicle-treated mouse (top) (original magnification � 400). The ratio of positive cells was calculated(number of positive synoviocytes/total synoviocytes counted) and is presented as the percentage (bottom). Values are the mean and SD of 7 miceper group. � � P � 0.001. E, Plasma concentration of SFN at various time points after intraperitoneal injection of 7.5 mM SFN, as determined byliquid chromatography mass spectrometry. Values are the mean � SD of 3 mice at each time point.
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stimulation with CII, but not with medium alone (n � 12mice per group) (Figures 5C and D). A decrease in theseverity of clinical arthritis following treatment withSFN was similarly reproduced in these 2 groups (datanot shown). Interestingly, mice treated with SFN showedsignificant increases in the secretion of IL-10, an antiin-flammatory cytokine, by lymph node cells and spleencells (Figures 5C and D), indicating that SFN regulationof cytokine production is not due to a nonspecificcytotoxic effect on mononuclear cells. Taken together,our data suggest that the systemic administration of SFNto arthritic mice prevents a shift toward a Th17 responseand suppresses autoimmune responses to CII in vivo,and thereby ameliorates the clinical and pathologicseverity of arthritis.
Arthritis suppression by SFN treatment in apassive model of arthritis in mice. CIA is mediated bythe synergistic actions of both CII-reactive T cells andantibodies to CII (30). Since SFN inhibited CII-reactiveT cell activation and proliferation, it was unclearwhether SFN can inhibit synoviocyte hyperplasia inde-pendently of its action on adaptive immunity. To addressthis issue, we investigated the effects of SFN on a passivemodel of arthritis. As shown in Figures 6A and C, theseverity of arthritis induced by the injection of anti-CIIantibody was decreased by the intraperitoneal adminis-tration of SFN (1.5 mM). Peak arthritis severity occurredon day 10, and the peak arthritis severity score was10.3 � 2.8 (mean � SD) in vehicle-treated mice and5.5 � 2.1 in SFN-treated mice (P � 0.001).
Figure 5. Sulforaphane (SFN) modulation of autoimmune and cytokine responses in micewith collagen-induced arthritis (CIA). A, Levels of IgG antibody (Ab) to type II collagen(anti-CII) in mice treated with vehicle alone (n � 12) or with 1.5 mM (n � 10) or 7.5 mM(n � 8) SFN. Values are the mean and SD arbitrary units (AU). � � P � 0.05 versus 7.5 mMSFN–treated mice. B, Proliferative responses of lymph node and spleen cells from mice withCIA to CII. Pooled lymph node or spleen cells from 5 mice were cultured with or without100 �g/ml of CII, and the stimulation index was determined. The mean � SD backgroundcpm without CII was 14,823 � 2,092 for lymph node cells and 3,541 � 521 for spleen cells.Values are the mean and SD of 3 independent experiments, each performed in triplicate. �
� P � 0.05 versus vehicle-treated mice. C and D, Effects of SFN on cytokine production bylymph node cells and spleen cells from mice with CIA. Pooled lymph node (C) or spleen (D)cells from 6 mice were incubated with SFN or vehicle for 48 hours in the presence or absenceof 100 �g/ml of CII, and the levels of interleukin-17 (IL-17), tumor necrosis factor �(TNF�), IL-6, interferon-� (IFN�), and IL-10 in culture supernatants were measured byenzyme-linked immunosorbent assay. Data are representative of 2 independent experimentsthat yielded similar results, each of which was performed in triplicate. Values are the meanand SD. � � P � 0.05 versus vehicle-treated mice.
166 KONG ET AL
Paw swelling, as assessed by measuring the diam-eter of the arthritic ankles, also was lower in SFN-treated mice than in control mice (Figure 6B). More-over, compared with the vehicle-treated mice, the SFN-treated mice had a decreased degree of synovialhyperplasia and cartilage/bone destruction, as seen onthe histologic analysis (Figures 6D and E). Collectively,these data suggest that SFN is able to prevent thedevelopment of antibody-induced arthritis in mice andreduces the synoviocyte hyperplasia independently of itseffect on activated T cells.
DISCUSSION
RA synoviocytes are resistant to apoptosis andexhibit a transformed phenotype, which might be causedby chronic exposure to genotoxic stimuli, including re-active oxygen species and growth factors (13,17–19). Thefindings of the present study are the first to show thatSFN, an isothiocyanate derived from cruciferous vege-tables such as broccoli, strongly induced synoviocyteapoptosis in vitro. Moreover, the number of TUNEL�synoviocytes was significantly higher in SFN-treatedmice than in control mice. In parallel with these findings,addition of SFN to RA FLS resulted in a decrease in theexpression of Bcl-2 and pAkt, both of which are anti-apoptotic molecules, but increased the expression of Baxand p53, both of which are proapoptotic molecules.Given the apoptotic pathway noted in RA FLS (26), ourdata suggest that SFN promotes FLS apoptosis throughthe inactivation of pAkt, which may lead to alterations inthe expression of Bcl-2, p53, and Bax. In this regard,SFN may reduce RA inflammation by limiting theabnormal proliferation of RA synoviocytes.
The role of T cells in RA has been emphasized bystudies of IL-17, a proinflammatory cytokine (14–16,31).IL-17 is produced by activated CD4� andCD4�CD45RO� memory T cells (32–34), and it haspleiotropic effects that include the induction of TNF�,IL-1, IL-6, and IL-8 production from various types ofcells (31–37). IL-17 is produced in quantity by T cells inthe RA synovium, and it stimulates synovial fibroblaststo produce proinflammatory cytokines and matrix met-alloproteinases (35,36). In addition, IL-17 in RA syno-vial fluid is a potent stimulator of osteoclastogenesis(38), and IL-17 deficiency was found to completelyinhibit arthritis development in mice (39). Moreover, Tcell self-reactivity forms a cytokine milieu for the spon-taneous development of Th17 cells that cause auto-immune arthritis (39). Taken together, this evidenceindicates that antigen-specific T cells that produce IL-17are crucial to the pathogenesis of RA, and so, they arepotential treatment targets.
Our results demonstrated that SFN inhibits IL-17and TNF� production by RA T cells. The down-regulatory effects of SFN on IL-17 and TNF� produc-tion were confirmed in vivo. The precise in vivo mech-anisms for the inactivation of T cells by SFN remainunclear, but seem to differ according to the SFN dosage.In addition to its proapoptotic action on T cells at higherconcentrations, a nontoxic dose of SFN directly inhib-ited the activation of several transcription factors, suchas ROR�t and T-bet, which are required for the differ-
Figure 6. Sulforaphane (SFN) suppression of collagen antibody–induced arthritis (CAIA), a passively transferred arthritis. A, Decreasein the severity of CAIA by treatment with SFN. Anti–type II collagenantibody (Ab) was injected intravenously into vehicle-treated (n � 10)and SFN-treated (1.5 mM; n � 10) BALB/c mice, and 3 days later, 50�g of lipopolysaccharide was administered intraperitoneally. Themean arthritis index was calculated at the indicated time points. Valuesare the mean and SD. � � P � 0.001 versus vehicle-treated mice. B,Effect of SFN on paw edema in mice with CAIA, as determined by thepaw thickness index calculated at the indicated time points. Values arethe mean and SD. � � P � 0.05 versus vehicle-treated mice. C, Grossappearance of the front feet (top) and the hind feet (bottom) ofvehicle-treated and SFN-treated mice. D, Hematoxylin and eosinstaining of ankle joint sections from vehicle-treated and SFN-treatedmice. The ankle joint from the vehicle-treated control mouse (a and b)shows marked inflammation, erosion, and destructive arthritis of thetarsal joints. In contrast, the ankle joint from the SFN-treated mouse(c and d) shows no arthritis. Boxed areas in a and c are shown at highermagnification in b and d, respectively (original magnification � 40 ina and c; � 100 in b and d.) E, Histology score for the degree ofinflammation and synovial hyperplasia in mice with CAIA treated withvehicle (open bars) or with 1.5 mM SFN (solid bars). Values are themean and SD of 7 mice per group. � � P � 0.05; �� � P � 0.001,versus vehicle-treated mice.
SFN REGULATION OF RHEUMATOID INFLAMMATION 167
entiation of naive T cells into Th17 and Th1 cells,respectively. In addition, consistent with the findings ofprevious reports (11,40), SFN at lower concentrationsarrested cell-cycle progression in the G1 phase in RA Tcells (data not shown), which may lead to a furtherdecrease in IL-17 and TNF� production. Therefore,SFN might abolish the pathologic actions of RA T cellsin various ways in vivo, such as via apoptosis induction,transcriptional repression of Th17 cell differentiation,and inhibition of cell proliferation and cytokine produc-tion. These scenarios may occur independently of oneanother, but the one that plays the dominant role in thetarget tissues remains to be determined.
It has been documented that SFN shows directantiinflammatory activity at various conditions (41–45).For example, SFN was shown to inhibit inducible nitricoxide synthetase and cyclooxygenase 2 in IFN�-stimulated mouse macrophages (42). NF-�B appears tobe a molecular target for the SFN-mediated antiinflam-matory effects in macrophages (41). In addition, SFNpretreatment inhibited IL-8 production by airway epi-thelial cells stimulated with diesel extract (43). Rats fedbroccoli sprouts, which contain high levels of glucora-phanin that is metabolized to SFN, showed decreasedoxidative stress and inflammation in the kidneys and inthe cardiovascular system (44). Talalay et al (45) haverecently found that an extract of broccoli sprouts pro-tects human skin from the inflammation caused byexposure to ultraviolet light.
The present study is the first to demonstrate thatSFN suppresses chronic autoimmune arthritis in vivo.Immune responses to autoantigens, such as anti-CIIantibody, and the T cell response to CII were alsoreduced. In addition, SFN-treated mice showed amarked reduction in the production of the proinflam-matory cytokines IL-17, TNF�, IL-6, and IFN� by lymphnode cells and spleen cells stimulated with CII and acommensurate reduction in synovial hyperplasia. More-over, the synovial hyperplasia in mice with CAIA, apassive model of arthritis that is independent of adaptiveimmunity, was also alleviated by SFN injection. All ofthese data are consistent with our in vitro observationsconcerning the down-regulatory effect of SFN on syno-viocyte hyperplasia, T cell proliferation, and cytokineproduction, suggesting that SFN inhibits the severityof arthritis by inhibiting the autoreactive lymphocytesthat respond to CII as well as by blocking synoviocytehyperplasia.
When taking the findings of previous reports intoconsideration (41,42), it also appears that SFN maydirectly affect the activated macrophages in mice with
CIA and block the secretion of proinflammatory medi-ators. Collectively, in view of the various actions of SFNin vitro (3,41–45), its ultimate effects in vivo are likely tobe determined by a combination of antiinflammatory,antioxidant, proapoptotic, and antiangiogenic mecha-nisms.
It is unclear how great a concentration of SFN inarthritic joints is required to suppress rheumatoid in-flammation. After a single intraperitoneal injection of7.5 mM SFN, the mean plasma concentrations peaked at�210 �M at 1 hour, which is greater than the effectivedose of SFN we tested in vitro. Since an antiarthriticactivity of SFN was maximally evident at a dose of 7.5mM, the therapeutic dose can be determined to be belowthis level (�63.8 mg/kg). Of special note, no side effectswere seen throughout the experiments. Given the highefficacy of SFN in arthritic mice, we believe that thisagent may be viewed as a potential agent for alleviatingchronic autoimmune arthritis. Nevertheless, we shouldemphasize that our data do not imply that dietarysupplementation with SFN, such as broccoli extracts,could be used to treat RA. Neither the toxicity profilenor the pharmacokinetic data for SFN in humans areavailable. Thus, additional investigations of the safetyand efficacy of SFN in RA patients are warranted.
In summary, our study demonstrates that SFNinhibits synovial hyperplasia, activated T cell prolifera-tion, and the production of IL-17 and TNF� by RA Tcells. Moreover, SFN administration to mice with exper-imental arthritis suppressed arthritis severity, antigen-specific T cell proliferation, autoantibody production,and proinflammatory cytokine production. These find-ings suggest that the SFN may offer a new therapeuticapproach for the treatment of chronic autoimmunearthritis.
ACKNOWLEDGMENTS
We thank all of the members of the Institute of Boneand Joint Diseases at the Catholic University of Korea. Wealso thank Dr. Richard Bucala (Yale University School ofMedicine, New Haven, CT) for his review of the manuscript.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Wan-Uk Kim had full access toall of the data in the study and takes responsibility for the integrity ofthe data and the accuracy of the data analysis.Study conception and design. Kong, Hyun Ah Kim, Chung, Wan-UkKim.Acquisition of data. Kong, Yoo, Hyun-Sook Kim, Yea, Ryu.Analysis and interpretation of data. Yoo, Ryu, Chung, Cho, Wan-UkKim.
168 KONG ET AL
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