Effects of a novel tylophorine analog on collagen-induced arthritis through inhibition of the innate immune response

ARTHRITIS & RHEUMATISMVol. 54, No. 3, March 2006, pp 877–886DOI 10.1002/art.21640© 2006, American College of Rheumatology

Effects of a Novel Tylophorine Analog onCollagen-Induced Arthritis Through Inhibition of the

Innate Immune Response

Xin You,1 Meng Pan,1 Wenli Gao,1 Her-Shyong Shiah,1 Jian Tao,1 Dongqing Zhang,1

Fotios Koumpouras,1 Shuang Wang,1 Hongyu Zhao,1 Joseph A. Madri,1 David Baker,2

Yung-Chi Cheng,1 and Zhinan Yin1

Objective. To test the effects of a novel tylopho-rine analog, DCB 3503, on the prevention and treatmentof collagen-induced arthritis (CIA) and to elucidate itsunderlying mechanisms.

Methods. DBA/1J mice were immunized with typeII collagen, and in some cases, lipopolysaccharide (LPS)was used to boost the development of arthritis. DCB3503 was injected intraperitoneally before or after theonset of CIA. Mice were monitored to assess the effectsof DCB 3503 on the clinical severity of the disease, andpathologic changes in the joints were examined histo-logically. Levels of tumor necrosis factor � (TNF�) andinterleukin-1� (IL-1�) in serum and joint tissues weremeasured by enzyme-linked immunosorbent assay andby cytometric bead array analysis. The effect of DCB3503 on LPS-induced proinflammatory cytokines frombone marrow–derived dendritic cells was determined byflow cytometry.

Results. DCB 3503 significantly suppressed the

development and progression of CIA. Moreover, DCB3503 completely blocked the LPS-triggered accelerationof joint inflammation and destruction. Consistent withits effects in vivo, DCB 3503 significantly suppressed thesynthesis of proinflammatory cytokines in inflamedjoints as well as cytokine synthesis by macrophages exam-ined ex vivo. Treatment also reduced the levels of inflam-matory cytokines (IL-6, IL-12, TNF�, and monocyte che-motactic protein 1) produced by bone marrow–deriveddendritic cells in vitro. However, DCB 3503 showed nodirect effects on T cell proliferation and B cell antibodyresponse.

Conclusion. Because of its ability to specificallysuppress innate immune responses, DCB 3503 may be anovel therapeutic agent for inflammatory arthritis inhumans.

Rheumatoid arthritis (RA) is a common systemicautoimmune disease that leads to joint inflammationand progressive cartilage and bone erosion (1–3). Al-though current treatments that specifically block a singlecytokine, such as anti–tumor necrosis factor � (anti-TNF�) or anti–interleukin-1 (anti–IL-1), have provedclinically effective, such treatments are often insufficientto produce complete disease remission in some patients(4,5). Consequently, there has been a great demand fornew antirheumatic agents that are able to act on multi-ple cytokines or mediators of inflammation, but havefewer toxic effects. There are effective treatments forrheumatism available from practitioners of traditionalChinese medicine, including the medicinal plant Tylo-phora atrofolliculata, from the family Asclepiadaceae (6).Thus, studies exploring the herbs of traditional Chinesemedicine may ultimately provide additional therapeuticagents for treating this chronic inflammatory disease.

Dr. Yin’s work was supported in part by an Arthritis Foun-dation Investigator award and by NIH grants K01-AR-02188 and1-R01-AI-056219.

1Xin You, MD, PhD (current address: University of Califor-nia, San Diego), Meng Pan, MD (current address: Ruijing Hospital,Shanghai Second Medical University, Shanghai, China), Wenli Gao,MD, PhD, Her-Shyong Shiah, MD, Jian Tao, PhD, Dongqing Zhang,MD, PhD (current address: Shanghai Jiao Torg University, Shanghai,China), Fotios Koumpouras, MD, Shuang Wang, PhD (current ad-dress: Columbia University, New York, New York), Hongyu Zhao,PhD, Joseph A. Madri, MD, PhD, Yung-Chi Cheng, PhD, Zhinan Yin,MD, PhD: Yale University, New Haven, Connecticut; 2David Baker,PhD: University of Tennessee, Knoxville.

Drs. You and Pan contributed equally to this work.Address correspondence and reprint requests to Zhinan

Yin, MD, PhD, Section of Rheumatology, Yale University Schoolof Medicine, Box 208031, The Anylan Center (TAC), Room 517, 300Cedar Street, New Haven, CT 06520-8031. E-mail: [email protected].

Submitted for publication August 3, 2005; accepted in revisedform November 10, 2005.

877

Collagen-induced arthritis (CIA) is a well-studiedanimal model of RA (7). Although the precise mecha-nisms whereby immunization with type II collagen (CII)leads to the development of chronic arthritis are notknown, there is considerable evidence implicating CII-specific CD4� T cells as primary mediators of diseaseinduction, with the production of complement-fixinganti-CII antibody being the major immune mechanismfor the initiation of joint inflammation (8–10). In thejoints, activated T cells and Th1-like cytokines inducemacrophages to produce TNF�, which in turn, leads toinfiltration by other inflammatory cells. These cells thenrelease other cytokines (IL-1 and IL-6) and mediators ofinflammation (inducible nitric oxide synthase and cyclo-oxygenase 2), resulting in persistent inflammation andjoint destruction (3,8,11). The CIA model has proveduseful in the development of new therapies for RA, suchas anti-TNF� and anti–IL-1 (12).

Several alkaloid extracts have recently been iso-lated and synthesized from Tylophora species. One syn-thesized tylophorine analog, DCB 3503 (NSC-716802),has been shown to selectively inhibit TNF�-inducedNF-�B activity and to suppress the growth of humantumor xenografts in nude mice (13). Since NF-�B ispivotal in joint inflammation, we hypothesized that DCB3503 might also be beneficial in the treatment of inflam-matory arthritis. To test our hypothesis, we used theDBA/1J (H-2q) mouse model of arthritis (14) and ad-ministered DCB 3503 at different time points postim-munization, either before or after the onset of CIA. Weshow herein that DCB 3503 significantly suppressedjoint inflammation and destruction, especially in the caseof lipopolysaccharide (LPS)–triggered acceleration ofCIA. Furthermore, DCB 3503 inhibited the productionof multiple mediators of inflammation ex vivo as well asby cultured dendritic cells in vitro, with little effect on Tcells and B cells.

MATERIALS AND METHODS

Mice. Male DBA/1J mice were purchased from TheJackson Laboratory (Bar Harbor, ME) and maintained underspecific pathogen–free conditions at the Yale UniversitySchool of Medicine. All animal procedures were performedwith the approval of the Institutional Animal Care and UseCommittee of the Yale University School of Medicine.

Chemicals, antibodies, and cytokines. Tylophorine an-alog DCB 3503 (NSC-716802) was synthesized in the labora-tory of one of us (DB) (13). Antibodies for flow cytometry andenzyme-linked immunosorbent assay (ELISA) were purchasedfrom BD PharMingen (San Diego, CA), except where indi-cated otherwise. Recombinant cytokines used for T cell acti-

vation and dendritic cell differentiation were purchased fromR&D Systems (Minneapolis, MN), except where indicatedotherwise.

CIA model and DCB 3503 treatment. The CIA modelwas adapted as described previously (14). On day 0, maleDBA/1J mice ages 8–10 weeks were injected at the base of thetail with bovine CII (Chondrex, Redmond, WA) emulsified inequal volumes of Freund’s complete adjuvant (CFA) (contain-ing 2 mg/ml of inactivated Mycobacterium tuberculosis; Chon-drex). On day 21, a booster injection with an emulsion of 100�g of bovine CII dissolved in Freund’s incomplete adjuvant(IFA; Chondrex) was given. Thereafter, mice were monitoreddaily for signs of arthritis. Assessors had no knowledge of thegroup to which the mice belonged. Arthritis severity wasgraded as described previously (15). Briefly, each paw wasscored individually on a scale of 0–3, where 0 � normal; 1 �mild/moderate, but definitely visible, swelling of 1 or moredigits; 2 � severe erythema and swelling affecting an entirepaw or joint; 3 � deformed paw or joint, with ankylosis andrigidity (significantly reduced hock joint motion on flexion/extension). Scores for all 4 paws were summed for each mouse(maximum score 12).

For the LPS-enhanced CIA model, LPS was injectedintraperitoneally (50 �g/mouse) on day 28 postimmunization,as previously described (16).

To test the effects of DCB 3503 on CIA, mice weretreated with either DCB 3503, or vehicle control at differenttime points postimmunization. Intraperitoneal injections ofDCB 3503 or vehicle were administered at a dosage of 6 mg/kgof body weight twice daily every 3 days. This injection schedulewas determined from a schedule-optimizing animal study usinghuman pancreatic cancer PANC-1 xenografts in nude mice.Our results showed that treatment with DCB 3503 twice dailyon every third day resulted in minimal toxicities in terms of lossof body weight and decrease in blood cell counts (red bloodcells, white blood cells, and platelets).

Based upon the kinetics of onset of CIA in this model,the mice were treated either before the onset of inflammation(early treatment) or after the onset of inflammation (latetreatment). In the early treatment cohort, mice were immu-nized with CII/CFA and on day 21 postimmunization, wererandomly divided into 2 groups and administered DCB 3503 orvehicle just prior to their booster immunization (CII/IFA).Treatment continued until day 30 (twice daily every 3 days). Inthe late treatment cohort, mice were immunized with CII/CFA, and on day 29 postimmunization, those with arthritis(score of 2–7) were divided into 2 equal cohorts and adminis-tered DCB 3503 or vehicle for a period of 13 days (twice dailyevery 3 days).

To define the effects of DCB 3503 on the LPS-enhanced CIA model, mice with arthritis scores of �1 wereinjected intraperitoneally with LPS (50 �g/mouse). Mice werethen treated either 24 hours before (early) or 48 hours after(late) LPS challenge. The severity of arthritis was monitoreddaily and scored as described above.

Cell preparation and culture. Bone marrow–deriveddendritic cells were generated using granulocyte–macrophagecolony-stimulating factor for 7 days, as described previously(17,18). Bone marrow–derived dendritic cells were activatedwith different concentrations of LPS (10 ng/ml to 1 �g/ml) or

878 YOU ET AL

poly(I-C) (1–10 �g/ml) in the presence or absence of DCB3503 for 6 or 24 hours, with the addition of brefeldin A for thelast 3 hours, and cells were used for intracellular cytokine(IL-12 and TNF�) staining. Supernatants were also collectedfor analysis of cytokine levels.

Cytokine detection. Cytokine levels were determinedby the following 3 methods. For cytokine determinations incultured cells or macrophages ex vivo, intracellular cytokinestaining was used as described in our previous studies (19). Forcytokine determinations in culture supernatants or serum,cytometric bead array kits (BD Biosciences, San Diego, CA)were used as described previously (20). For cytokine determi-nations in joints, ankle joints were removed, frozen in liquidnitrogen, smashed, mixed with 200 �l of phosphate bufferedsaline (PBS) at 4°C, centrifuged at 10,000 revolutions perminute, and the supernatants were assayed by ELISA (forIL-1�; R&D Systems) and by cytometric bead array (for IL-12,TNF�, interferon-� [IFN�], IL-6, IL-10 and monocyte chemo-tactic protein 1 [MCP-1]).

T cell proliferation assay. Single-cell suspensions de-rived from the spleen were prepared and cultured as describedpreviously (19). CD4� T cells were negatively selected fromsplenocytes of mice that had been immunized and treated witheither DCB 3503 or vehicle, as described previously (14). Thepurity of CD4� T cells was �95%, and similar percentages ofactivated CD4� T cells (CD62Llow,CD44high) were detected inthese 2 groups of mice (data not shown). T cell–depletedsplenocytes from DBA/1J mice were treated with mitomycin C(Sigma-Aldrich, St. Louis, MO) for 45 minutes (0.5 �g/ml) andused as antigen-presenting cells. CD4� T cells (1 � 106/ml)were incubated together with antigen-presenting cells (5 �106/ml) in the presence of different concentration of T cell–

grade bovine CII for 72 hours, and pulsed with 3H-thymidine(20 �Ci/well) for 18 hours before harvesting. Uptake ofradiolabeled thymidine (in counts per minute) was measuredin a liquid scintillation counter as described elsewhere (14).

ELISA for serum anti-CII antibodies and their iso-types. Serum anti-CII antibodies and their isotypes weremeasured by ELISA, as described previously (14). Briefly,flat-bottomed 96-well ELISA plates were coated overnight at4°C with bovine CII (0.5 �g/well). After blocking the plateswith 5% fetal bovine serum–PBS, mouse sera were diluted1:800, added to duplicate wells, and incubated for 2 hours atroom temperature. Plates were washed, and biotin-conjugatedgoat anti-mouse IgG, IgG1, and IgG2a were added at adilution of 1:2,000 for 1 hour, followed by the addition ofstreptavidin–horseradish peroxidase (Sigma-Aldrich). Plateswere then developed and antibody levels measured as de-scribed previously (14).

Histopathologic assessment. Mice were killed andjoints were removed, immediately fixed in 10% bufferedformalin, and decalcified in a decalcifying solution for 3–5days. The tissue was then processed and embedded in paraffin.Five-micrometer tissue sections were prepared and stainedwith hematoxylin and eosin using standard methods. All sec-tions were stained by the Yale Pathology Laboratory and readblindly by a pathologist (JAM).

Statistical analysis. Analyses of arthritis severity datawere performed using SAS version 8.1 software (SAS Institute,Cary, NC). Statistical testing was 2-sided at a 5% level ofsignificance. Exploratory data analysis was performed by as-sessing descriptive statistics (mean and SD) for each variableusing SAS PROC Univariate and PROC Freq programs. The

Figure 1. Blocking of the development and progression of collagen-induced arthritis (CIA) by treatmentwith DCB 3503. CIA was induced in 8-week-old male DBA/1J mice by immunization with type II collagen(CII) as described in Materials and Methods. DCB 3503 was administered by intraperitoneal injection ata dose of 6 mg/kg at different time points postimmunization, and the incidence and severity of arthritiswere recorded daily. A, Early treatment with DCB 3503 significantly reduced the severity of arthritis anddelayed its onset. On day 21 postimmunization, CII-immunized mice were divided into 2 groups and weretreated with DCB 3503 (n � 8) or with vehicle (n � 10) from day 21 to day 30. Arthritis progression wasfollowed until day 42. B, Late treatment with DCB 3503 significantly inhibited arthritis progression. Onday 29 postimmunization, CII-immunized arthritic mice were divided into 2 groups and treated with DCB3503 (n � 15) or with vehicle (n � 14) until day 41. Arthritis severity was assessed until day 41. Valuesare the mean and SD. � � P � 0.0001 as determined with SAS version 8.1 software.

EFFECTS OF A NOVEL TYLOPHORINE ANALOG ON CIA 879

study variable was arthritis scores over time. Outcomes wereassessed for normality prior to analysis, using normal proba-bility plots and Kolmogorov-Smirnov test statistics. Arthritisscores were analyzed using the PROC Mixed program andmixed effects models, with treatment group, time, and treat-ment group by time interaction as the fixed effects and astructured variance–covariance pattern matrix. The best-fittingvariance–covariance matrix according to the Bayesian infor-mation criterion was selected. Cytokine levels were comparedusing Student’s unpaired 2-tailed t-test or nonparametric ana-lysis (Wilcoxon’s or Mann-Whitney test) if the SDs weresignificantly different between the 2 groups being compared,using InStat 2.03 software (for Macintosh; GraphPad, SanDiego, CA).

RESULTS

Significant blocking of the development and pro-gression of CIA by treatment with DCB 3503. To definethe therapeutic effects of DCB 3503 on CIA in DBA/1Jmice, we used 2 different treatment protocols, early andlate treatment (before and after the onset of CIA). Inthe early treatment cohort, DCB 3503 significantly de-creased the severity and delayed the onset of arthritis(Figure 1A). On day 30, only 1 of the 8 mice in the activetreatment group (12.5%) developed a mild arthritis,whereas 9 mice of the 10 mice in the vehicle treatmentgroup (90%) developed arthritis (mean score �3.0)(P � 0.0001). It should be noted that upon the termina-tion of therapy, the incidence and severity of arthritisincreased in the treated group; however, 10 days aftertreatment termination, the mean � SD arthritis scoreswere still significantly lower than those in the controlgroup (2.75 � 2.8 versus 4.6 � 2.2). Thus, DCB 3503significantly blocked the development of arthritis anddelayed its onset.

In the late treatment cohort, mice treated withvehicle continued to develop severe arthritis, whereasmice treated with DCB 3503 showed no progression or areduced severity of arthritis (P � 0.0001) (Figure 1B).Thus, our results suggest that DCB 3503 can blockthe development and progression of inflammatory ar-thritis.

Significant suppression of LPS-triggered jointinflammation by treatment with DCB 3503. To amplifyand equalize the degree of arthritis in mice with CIA,animals were injected with LPS, as previously described(16) and then treated with DCB 3503 or vehicle accord-ing to 2 difference protocols, early and late treatment(1 day before or 2 days after LPS injection). In theearly treatment cohort, all 5 mice treated with vehicledeveloped severe arthritis, with marked swelling anderythema of the hind paws and fore paws (Figure 2A).

Inflammation in these mice affected the ankle jointsand extended distally through the limb and the digits.The maximum arthritis score was 12, with mean score

Figure 2. Histopathologic features of representative ankle joints har-vested from male DBA/1J mice with collagen-induced arthritis (CIA). A,Control mouse on day 35 after CIA induction, showing robust pannusformation (P) and significant articular cartilage erosion (boxed area in toppanel). High-power view of the boxed area (bottom) shows pannuseroding into and replacing the articular cartilage (C) overlying bone (B).B, DCB 3503–treated mouse on day 35 after disease induction, showingwell-preserved joint spaces (JS) and articular cartilage surfaces (boxedarea in top panel), with minimal pannus formation. High-power view ofthe boxed area (bottom) shows well-preserved articular cartilage and anopen, well-preserved joint space. C, Control mouse on day 50 after diseaseinduction, showing more extensive pannus formation and bone andcartilage destruction (boxed area in top panel) compared with day 35.High-power view of the boxed area (bottom) shows pannus formation,with extensive articular cartilage destruction. An articular cartilage rem-nant (CR) is surrounded by the invading pannus. D, DCB 3503–treatedmouse on day 50 after disease induction, showing a normal joint space andwell-preserved articular cartilage (boxed area in top panel). High-powerview of the boxed area (bottom) shows normal-appearing articularcartilage and an open joint space. (Original magnification � 10; � 40 inhigh-power views.)

880 YOU ET AL

of 10. In contrast, the group treated with DCB 3503was still free of arthritis after LPS challenge; only 2mice had mild arthritis, with scores of �2 (P � 0.001)(Figure 3A).

In the late-treatment group, upon LPS injection,mice with a mean arthritis score of 5 were divided into 2groups and treated with either DCB 3503 or vehiclefrom day 30 to day 42 postimmunization. Mice in thevehicle control group developed severe arthritis, whichpeaked on day 35 with a mean arthritis score of 11. Micein the active treatment group showed no signs of pro-gression and maintained similar arthritis scores as forthe prior treatment (P � 0.0001) (Figure 3B). Ourresults therefore demonstrate that LPS-triggered inflam-matory responses can be blocked by administering DCB3503.

Findings of histopathologic analysis. Furtherevidence to support the inhibitory effects of DCB 3503on LPS-accelerated arthritis was obtained by histo-pathology analysis. On day 35, control mice exhibitedrobust pannus formation and significant articularcartilage erosion, with pannus eroding into and re-placing the articular cartilage overlying the bone(Figure 2A). In contrast, DCB 3503–treated mice exhib-

ited well-preserved joint spaces and articular cartilagesurfaces, with minimal pannus formation (Figure 2B).On day 50, control mice exhibited more extensive pan-nus formation and destruction of bone and cartilage(Figure 2C) as compared with control joints on day 35.Figure 2C shows an articular cartilage remnant sur-rounded by the invading pannus. In contrast, DCB3503–treated mice showed a normal joint space andwell-preserved articular cartilage (Figure 2D), indicatingthat the administration of DCB 3503 directly correlatedwith a reduction in disease severity. Similar effects onjoint pathology were seen in DCB 3503–treated micewith CIA (data not shown).

Suppression of the production of proinflamma-tory cytokines ex vivo by treatment with DCB 3503. Todefine the underlying mechanisms of the effect of DCB3503, we examined the systemic and local levels of thesecytokines ex vivo. Treatment with DCB 3503 before theonset of arthritis significantly reduced the level of TNF�in serum (Figure 4A). The level of IFN� in the samegroups of serum samples was also reduced, whereas thelevel of IL-4 was increased, although the difference wasnot statistically significant (data not shown).

To test the effect of DCB 3503 on LPS-triggered

Figure 3. Blocking of joint inflammation and progression of lipopolysaccharide (LPS)–acceleratedcollagen-induced arthritis (CIA) by treatment with DCB 3503. Male DBA/1J mice immunized with typeII collagen (CII) were given an intraperitoneal injection of LPS (50 �g/mouse) on day 28 postimmuni-zation to enhance arthritis severity. Mice were treated with DCB 3503 or vehicle either 1 day before(early treatment) or 1 day after (late treatment) LPS injection. A, Early treatment with DCB 3503completely blocked LPS-accelerated CIA. On day 28 postimmunization, CII-immunized mice witharthritis scores �1 were divided into 2 groups and were treated with either DCB 3503 or vehicle (n � 5per group), which was followed the next day (day 29) by LPS injection. The development of arthritis wasmonitored and recorded. B, Late treatment with DCB 3503 slowed the progression of LPS-acceleratedCIA. On day 28 postimmunization, CII-immunized mice with arthritis scores �1 were challenged withLPS (50 �g/mouse injected intraperitoneally), and 2 days later (day 30), mice with arthritis scores of 3–7were divided into 2 groups and were treated with DCB 3503 or vehicle (n � 6 per group) from day 30to day 42 postimmunization. Values are the mean and SD. � � P � 0.0001 as determined with SASversion 8.1 software.


Figure 4. Blocking of the production of tumor necrosis factor � (TNF�) and interleukin-1� (IL-1�) ex vivo by treatment with DCB 3503. A, DCB3503 treatment reduced serum levels of TNF� in male DBA/1J mice. Levels of TNF� were measured by cytometric bead array in sera from micewith collagen-induced arthritis (CIA) on days 28 and 35 postimmunization, as described in Figure 3A. Values are the mean and SD (n � 5 pergroup). �� P � 0.01 by student’s t-test. B, DCB 3503 blocked the production of TNF� by macrophages ex vivo. Spleen cells were obtained on day35 postimmunization from mice with lipopolysaccharide (LPS)–triggered CIA and directly stained for intracellular TNF�. Shown are 2representative samples from each group. Numbers in the right upper quadrants represent percentages of CD11b�,TNF�� cells. C, DCB 3503completely blocked IL-1� production in ankle joints. Joint tissues were obtained on day 35 postimmunization, as described in Figure 3A, fromarthritic mice with LPS-triggered CIA, treated with vehicle or DCB 3503, and homogenized in 200 �l of phosphate buffered saline. Levels of IL-1�in the supernatants were measured by enzyme-linked immunosorbent assay. Values are the mean and SD (n � 3 per group). � � P � 0.001 bystudent’s t-test. D, Treatment with DCB 3503 inhibited the production of proinflammatory cytokines and mediators of inflammation by bonemarrow–derived dendritic cells. Cells were pretreated with DCB 3503 or vehicle for 1 hour and then activated with LPS (1 �g/ml) or poly(I-C) (5�g/ml). After 6 hours of culture, cells were fixed and stained for intracellular IL-12 and TNF�. Shown are the results of a representative experiment.

882 YOU ET AL

TNF� production by macrophages ex vivo, splenocyteswere obtained from mice with LPS-triggered CIA on day35 postimmunization and were directly stained withintracellular TNF�. Mice treated with DCB 3503 pro-duced significantly less TNF� than those treated withvehicle (Figure 4B).

Local levels of IL-1� were measured in jointtissues from mice with LPS-triggered CIA on day 35postimmunization. Tissues were homogenized andwashed with 200 �l of PBS per joint, and cytokine levelsin the supernatant were determining by ELISA andcytometric bead array. DCB 3503 essentially blocked theproduction of IL-1� (Figure 4C), as well as severalother inflammatory cytokines (TNF�, IL-6, IL-12, andMCP-1) (Table 1), at sites of local inflammation. Takentogether, these data strongly suggest that DCB 3503suppresses the development of inflammatory arthritisand joint destruction through blocking the productionof inflammatory cytokines such as TNF�, IL-1�, andothers.

Suppression of the production of mediators ofinflammation by bone marrow–derived dendritic cellsby treatment with DCB 3503. Based upon the effects ofDCB 3503 on LPS-triggered joint inflammation anddestruction, we next tested the effects of this compoundon the production of proinflammatory cytokines andmediators of inflammation by bone marrow–deriveddendritic cells. Bone marrow–derived dendritic cellswere pretreated with either vehicle or different concen-trations of DCB 3503 for 1 hour, and cells were thenactivated with LPS (1 �g/ml) or poly(I-C) (5 �g/ml) for24 hours, with the addition of brefeldin A for the last 3

hours. These cells were then used for intracellularcytokine staining.

DCB 3503 significantly suppressed both the LPS-triggered and the poly(I-C)–triggered production ofIL-12 and TNF�, especially IL-12�,TNF�� double-positive cells (from 12.4% to 1.73% for LPS, and from50% to 5% for poly[I-C]) in a concentration-dependentmanner (Figure 4D). Similar results were obtained withbone marrow–derived macrophages or peritoneal macro-phages (data not shown). To confirm the results fromflow cytometry, supernatants from parallel cultures ac-tivated with LPS (1 �g/ml) for 24 hours were analyzed bycytometric bead array techniques (6 cytokine array kits,including IL-12 p75, TNF�, IFN�, IL-6, IL-10, andMCP-1). DCB 3503 significantly reduced proinflamma-tory cytokine production by 4–11-fold (Table 1), with noeffect on the secretion of IL-10 and IFN� analyzed inthe same array (data not shown).

Absence of direct effects of DCB 3503 on antigen-specific T and B cell functions. Both T cells and B cellshave been demonstrated to be essential for the initiationof inflammatory arthritis (1,8). To test the effects ofDCB 3503 on the CII-specific T cell proliferation re-sponse, DBA/1J mice were immunized with CII/CFAand treated either with DCB 3503 or vehicle on the dayof booster immunization (day 21). On day 28 postimmu-nization, the CD4� T cell population was enriched, andthe proliferative response of this T cell subset to CII wasmeasured by 3H-thymidine uptake assay. DCB 3503treatment showed little effect on the CII-specific T cellproliferative response (Figure 5A).

To test the effects of DCB 3503 on the function

Table 1. DCB 3503 suppression of proinflammatory cytokine production by ankle joint tissues and bonemarrow–derived dendritic cells*

TNF�,mean � SD pg/ml

IL-6,mean � SD pg/ml

IL-12,mean � SD pg/ml

MCP-1,mean � SD pg/ml

Joint tissueVehicle 760 � 47† 476 � 36† 190 � 26‡ 261 � 18†DCB 3503 68 � 28 68 � 29 48 � 35 51 � 15

Dendritic cellsVehicle 4,767 � 509† 4,659 � 386† 950 � 40† 651 � 33†DCB 3503 868 � 124 544 � 119 230 � 30 59 � 9

* Ankle joints were prepared and analyzed for cytokine content as described in Materials and Methods,using cytometric bead array proinflammatory cytokine kits (6 cytokine arrays). Bone marrow–deriveddendritic cells were generated as described in Materials and Methods. Equal numbers of cells werepretreated with vehicle or with DCB 3503 for 1 hour, followed by lipopolysaccharide activation (1 �g/ml) for24 hours, and the levels of proinflammatory cytokines were determined by cytometric bead array analysis.TNF� � tumor necrosis factor �; IL-6 � interleukin-6; MCP-1 � monocyte chemotactic protein 1.† P � 0.001 versus DCB 3503 treatment.‡ P � 0.01 versus DCB 3503 treatment.


of B cells, we examined by ELISA the levels of CII-specific antibodies and their isotypes in serum samplescollected at different time points postimmunizationfrom mice with CIA or with LPS-triggered CIA. Figure5B shows the results obtained in serum samples col-lected from LPS-triggered arthritic mice. On day 14, theanti-CII antibody (IgG1 and IgG2a) was readily detect-able in all immunized mice. The levels of anti-CIIantibodies peaked on day 28 postimmunization. DCB3503 treatment had no direct effect on the antibodyresponse to CII at all time points analyzed (Figure 5B).Similar results were obtained with serum samples frommice with CIA (data not shown). These results indicatethat DCB 3503 has no direct effect on either T cell or Bcell immune responses.

DISCUSSION

RA is a common autoimmune disease that resultsin progressive joint inflammation and destruction (1).Although therapy with anti-TNF� and anti–IL-1 pro-vides an alternative or supplemental approach to treat-ment with corticosteroids or methotrexate, the efficacyof these antagonists can be limited (12,21). Therefore, a

search for novel therapeutic agents is greatly needed notonly to improve outcomes, but also to reduce thefinancial burden of current antirheumatic regimens.Chinese herbal medicines, such as the Tylophora plant,have been used for the treatment of rheumatism for athousand years (6), and it is possible that such herbscould be used as an alternative approach to the treat-ment of RA, provided that the effective components areidentified and their toxicities are understood. A newlysynthesized tylophorine analog derived from Tylophoraspecies, DCB 3503, has recently been shown to havepotent effects against tumor growth in vitro as well as invivo by is effects on cAMP, activator protein 1 (AP-1), oron NF-�B–mediated transcription (13). This study is thefirst to demonstrate that DCB 3503 effectively inhibitsthe development and progression of CIA in DBA/1Jmice by suppressing the production of proinflammatorycytokines and mediators of inflammation by innateimmune cells.

We demonstrated that early treatment with DCB3503 significantly suppressed the development of arthri-tis, delayed its onset, and reduced its severity (Figure1A). More impressively, treatment with DCB 3503 com-

Figure 5. Lack of effect of DCB 3505 on T cell proliferation and antibody response in DBA/1J mice withcollagen-induced arthritis. A, DCB 3503 had no effect on type II collagen (CII)–specific T cellproliferation. Age-matched male DBA/1J mice were immunized with CII in Freund’s complete adjuvantand, on day 21, were given a booster injection of CII in Freund’s incomplete adjuvant; at that time, micewere also treated with either DCB 3503 or vehicle. On day 28 postimmunization, splenocytes enriched inCD4� T cells were analyzed for their proliferative response to CII. Shown are the results of arepresentative experiment of 3 separate experiments performed. B, DCB 3503 had no effect on theCII-specific antibody response. Antibody levels in serum samples from mice described in Figures 1 and 3were analyzed by enzyme-linked immunosorbent assay. Shown are the results for the mice described inFigure 3.

884 YOU ET AL

pletely blocked the accelerated arthritis triggered byLPS (Figure 3A), with a reduction in inflammatory cellinfiltration, prevention of pannus formation and carti-lage erosion, and preservation of the joint architecture(Figure 2). Late treatment with DCB 3503 (after theonset of CIA) suppressed the progression of arthritis(Figures 1B and 3B) and reduced the degree of jointdestruction (data not shown). Our results suggest thatthis tylophorine analog may potentially be used as atherapeutic agent for inflammatory arthritis in humans.

How does DCB 3503 block joint inflammationand destruction? The striking feature of DCB 3503 isthat it suppressed the production of multiple proinflam-matory cytokines and mediators of inflammation. Morespecifically, it reduced both systemic and local levels ofTNF� and IL-1� (Figure 4) and suppressed the ability ofinnate immune cells to produce multiple inflammatorycytokines (TNF�, IL-12, IL-6) and chemokine MCP-1(Figure 4 and Table 1), as well as other mediators ofinflammation (iNOS and COX-2) (data not shown). Theroles of these molecules in the pathogenesis of jointinflammation and destruction have been extensivelystudied (3,8,21,22). All of these molecules have beenshown to play a divergent role in the different processesof joint inflammation and destruction. Moreover, it hasbeen demonstrated that TNF� plays a major role in theinfiltration of inflammatory cells, whereas IL-1 has beenshown to be involved in the destruction of the cartilagematrix (23). These 2 cytokines can also trigger theactivation of NF-�B, which leads to the production ofadditional inflammatory cytokines that will activate acascade of events that cause joint inflammation anddamage (24,25). Both iNOS and COX-2 are importantmediators of IL-1–induced bone erosion and edema inthe CIA model (26,27).

Our results indicate a role of DCB 3503 ininhibiting both the inflammation and the destructioncaused by inflammatory arthritis, possibly through mul-tiple mechanisms. DCB 3503 has potent inhibitory ef-fects on AP-1 and NF-�B (13), transcription factors thatplay a critical role in the pathogenesis of RA in humansand CIA in mice (24,28,29). It is therefore reasonable tospeculate that DCB 3503 could suppress the develop-ment and progression of inflammatory arthritis by actingon these essential signaling pathways. Further studiesare needed to define the underlying molecular mecha-nisms that mediate the effects of DCB 3503 on thesesignaling pathways.

In contrast to its effects on innate immune cells,DCB 3503 had no direct effect on the function of T cellsor B cells, as evidenced by the fact that both the

CII-specific T cell proliferative response and anti-CIIantibody production were not altered (Figure 5). Al-though DCB 3503 significantly suppressed the produc-tion of proinflammatory cytokines, it is very likely thatDCB 3503 does not affect the ability of dendritic cells topresent antigen and prime the T cell response. Indeed,in our preliminary studies, DCB 3503 had little effect onLPS-triggered dendritic cell maturation (class II majorhistocompatibility complex and B71/B7-2 expression) invitro (data not shown). The fact that DCB 3503 attenu-ated joint inflammation and destructive arthritis withoutaffecting the adaptive immune response implies thatDCB 3503 has little or no effect on the initiation ofinflammatory joint disease, but rather, exerts its effectson downstream events that are important for the prop-agation of inflammatory joint disease.

It should be mentioned that in these studies, theadministration of DCB 3503 did not lead to a reductionin the body weight of the mice and did not change theperipheral blood cell counts (data not shown), and noobvious toxicity was observed. Similarly, no liver toxicitywas observed even after prolonged periods of adminis-tration of DCB 3503 in the MRL/lpr mouse model oflupus (Choi J-Y, Craft J: personal communication).Thus, DCB 3503 has the potential to be a safe agent inthe treatment of humans with RA.

In conclusion, we have discovered a new com-pound that selectively targets the production of proin-flammatory cytokines by innate immune cells. Thisresulted in striking attenuation of joint inflammationand joint destruction associated with arthritis in the CIAmouse model. This novel compound may have potentialin the treatment of humans with inflammatory arthritis.

ACKNOWLEDGMENTS

We thank Dr. Yunfei Gao and Dr. Insoo Kang forhelpful suggestions. We thank Dr. Bohdan Harvev for criticalreview of the manuscript.

REFERENCES

1. Feldmann M, Brennan FM, Maini RN. Rheumatoid arthritis. Cell1996;85:307–10.

2. Aho K, Koskenvuo M, Tuominen J, Kaprio J. Occurrence ofrheumatoid arthritis in a nationwide series of twins. J Rheumatol1986;13:899–902.

3. Feldmann M, Maini RN, Bondeson J, Taylor P, Foxwell BM,Brennan FM. Cytokine blockade in rheumatoid arthritis. Adv ExpMed Biol 2001;490:119–27.

4. Keystone EC, Kavanaugh A. What to do with TNF failures. ExpertOpin Drug Saf 2005;4:149–55.

5. Zink A, Listing J, Kary S, Ramlau P, Stoyanova-Scholz M,Babinsky K, et al. Treatment continuation in patients receiving


biological agents or conventional DMARD therapy. Ann RheumDis 2005;64:1274–9.

6. Yin Z, Siegert S, Neure L, Grolms M, Liu L, Eggens U, et al. Theelevated ratio of interferon �/interleukin-4-positive T cells foundin synovial fluid and synovial membrane of rheumatoid arthritispatients can be changed by interleukin-4 but not by interleukin-10or transforming growth factor �. Rheumatology (Oxford) 1999;38:1058–67.

7. Trentham DE, Townes AS, Kang AH. Autoimmunity to type IIcollagen an experimental model of arthritis. J Exp Med 1977;146:857–68.

8. Luross JA, Williams NA. The genetic and immunopathologicalprocesses underlying collagen-induced arthritis. Immunology2001;103:407–16.

9. Svensson L, Jirholt J, Holmdahl R, Jansson L. B cell-deficientmice do not develop type II collagen-induced arthritis (CIA). ClinExp Immunol 1998;111:521–6.

10. Yoshino S, Cleland LG, Mayrhofer G. Treatment of collagen-induced arthritis in rats with a monoclonal antibody against the�/� T cell antigen receptor. Arthritis Rheum 1991;34:1039–47.

11. Mauri C, Williams RO, Walmsley M, Feldmann M. Relationshipbetween Th1/Th2 cytokine patterns and the arthritogenic responsein collagen-induced arthritis. Eur J Immunol 1996;26:1511–8.

12. Feldmann M. Development of anti-TNF therapy for rheumatoidarthritis. Nat Rev Immunol 2002;2:364–71.

13. Gao W, Lam W, Zhong S, Kaczmarek C, Baker DC, Cheng YC.Novel mode of action of tylophorine analogs as antitumor com-pounds. Cancer Res 2004;64:678–88.

14. Pan M, Kang I, Craft J, Yin Z. Resistance to development ofcollagen-induced arthritis in C57BL/6 mice is due to a defect insecondary, but not in primary, immune response. J Clin Immunol2004;24:481–91.

15. Wang Y, Kristan J, Hao L, Lenkoski CS, Shen Y, Matis LA. A rolefor complement in antibody-mediated inflammation: C5-deficientDBA/1 mice are resistant to collagen-induced arthritis. J Immunol2000;164:4340–7.

16. Kang I, Lee WW, Lee Y. Modulation of collagen-induced arthritisby IL-4 and dexamethasone: the synergistic effect of IL-4 anddexamethasone on the resolution of CIA. Immunopharmacology2000;49:317–24.

17. Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N,et al. An advanced culture method for generating large quantitiesof highly pure dendritic cells from mouse bone marrow. J ImmunolMethods 1999;223:77–92.

18. Johnson CR, Kitz D, Little JR. A method for the derivation andcontinuous propagation of cloned murine bone marrow macro-phages. J Immunol Methods 1983;65:319–32.

19. Yin Z, Zhang DH, Welte T, Bahtiyar G, Jung S, Liu L, et al.Dominance of IL-12 over IL-4 in � delta T cell differentiationleads to default production of IFN-�: failure to down-regulateIL-12 receptor �2-chain expression. J Immunol 2000;164:3056–64.

20. Welte T, Zhang SS, Wang T, Zhang Z, Hesslein DG, Yin Z, et al.STAT3 deletion during hematopoiesis causes Crohn’s disease-likepathogenesis and lethality: a critical role of STAT3 in innateimmunity. Proc Natl Acad Sci U S A 2003;100:1879–84.

21. Abramson SB, Amin A. Blocking the effects of IL-1 in rheumatoidarthritis protects bone and cartilage. Rheumatology (Oxford)2002;41:972–80.

22. Dayer JM. The pivotal role of interleukin-1 in the clinical mani-festations of rheumatoid arthritis. Rheumatology (Oxford)2003;42 Suppl 2:ii3–10.

23. Joosten LA, Helsen MM, Saxne T, van de Loo FA, Heinegard D,van Den Berg WB. IL-1� � blockade prevents cartilage and bonedestruction in murine type II collagen-induced arthritis, whereasTNF-� blockade only ameliorates joint inflammation. J Immunol1999;163:5049–55.

24. Tak PP, Gerlag DM, Aupperle KR, van de Geest DA, OverbeekM, Bennet BL, et al. Inhibitor of nuclear factor �B kinase � is akey regulator of synovial inflammation. Arthritis Rheum 2001;44:1897–907.

25. Seetharaman R, Mora AL, Nabozny G, Boothby M, Chen J.Essential role of T cell NF-�B activation in collagen-inducedarthritis. J Immunol 1999;163:1577–83.

26. Van ’t Hof RJ, Armour KJ, Smith LM, Armour KE, Wei XQ, LiewFY, et al. Requirement of the inducible nitric oxide synthasepathway for IL-1-induced osteoclastic bone resorption. Proc NatlAcad Sci U S A 2000;97:7993–8.

27. Ochi T, Ohkubo Y, Mutoh S. Role of cyclooxygenase-2, but notcyclooxygenase-1, on type II collagen-induced arthritis in DBA/1Jmice. Biochem Pharmacol 2003;66:1055–60.

28. Han Z, Boyle DL, Manning AM, Firestein GS. AP-1 and NF-�Bregulation in rheumatoid arthritis and murine collagen-inducedarthritis. Autoimmunity 1998;28:197–208.

29. Miagkov AV, Kovalenko DV, Brown CE, Didsbury JR, CogswellJP, Stimpson SA, et al. NF-�B activation provides the potentiallink between inflammation and hyperplasia in the arthritic joint.Proc Natl Acad Sci U S A 1998;95:13859–64.

886 YOU ET AL

Documents

Effects of a novel tylophorine analog on collagen-induced arthritis through inhibition of the innate immune response