117

IIntroduction

ncreasing attention has been paid to the role ofproinflammatory cytokines, including interleukin-1,

interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα),in the pathogenesis of rheumatoid arthritis (RA).1,2 Agentsinhibiting these cytokines have been shown to be effectivein the treatment of RA.3,4 Thus, etanercept (a TNF-receptor-immunoglobulin G [IgG] constant fragment [Fc] fusionprotein) and infliximab (an anti-TNFα monoclonalantibody) suppress the inflammation of RA by inhibitingTNFα. Although several studies have disclosed the invitro effects of these biological agents on the immune-competent cells,5,6 their precise mechanisms of action inRA remain unclear.

We have recently demonstrated that etanercept andinfliximab suppressed the proliferation and interferon-γproduction of peripheral blood mononuclear cells(PBMCs) stimulated with staphylococcal enterotoxin B(SEB) without direct effects on immobilized anti-CD3-

　Original Contribution Kitasato Med J 2014; 44: 117-124　

Effects of TNF inhibitors on human monocytes

Toshihiro Tono, Satoko Aihara, Takayuki Hoshiyama, Yoshiyuki Arinuma,Tatsuo Nagai, Shunsei Hirohata

Department of Rheumatology and Infectious Diseases, Kitasato University School of Medicine

Objective: Our objective was to explore the effects of the tumor necrosis factor (TNF) inhibitors,etanercept and infliximab on the function of human monocytes.Methods: Monocytes from healthy donors were cultured in the presence of staphylococcal enterotoxinB (SEB) with pharmacologically attainable concentrations of biological agents or controlimmunoglobulin G (IgG). The expression of interleukin-6 (IL-6) mRNA was determined by real-timequantitative RT-PCR. The expressions of CD80 and CD86 and the induction of apoptosis of monocyteswere measured by flow cytometry.Results: Both etanercept and infliximab promoted apoptosis of SEB-stimulated monocytes. Theinduction of apoptosis of monocytes by these biological agents were reversed by the addition of IgGbut not by IgG F(ab')2 fragments. Etanercept and infliximab significantly suppressed the expressionsof CD80 and CD86 in SEB-stimulated monocytes as well as suppressing the expression of mRNA forIL-6 in SEB-stimulated monocytes.Conclusions: These results demonstrate that one of the mechanisms of action of TNF inhibitorsinvolves the induction of apoptosis of monocytes, which involves interaction with constant fragment(Fc) receptors on monocytes. Moreover, these data also indicate that TNF inhibitors strongly inhibitIL-6 production of monocytes and concurrently suppress the expression of costimulatory molecules.

Key words: monocytes, TNF inhibitors, apoptosis, mRNA, IL-6

stimulated CD4+ T cells.7 It was, therefore, suggestedthat etanercept and infliximab might have suppressedthe activation of PBMCs stimulated with SEB byinhibiting the function of monocytes.7

The present study, therefore, explores the effects ofTNF inhibitors on human monocytes in more detail.Special attention was paid to the capacities of TNFinhibitors to induce the apoptosis of monocytes and tomodulate the expression of costimulatory molecules andcytokines.

Materials and Methods

A variety of monoclonal antibodies (mAbs) were used inthis study, including fluorescein isothiocyanate (FITC)-conjugated anti-CD80 (Immunotech, Marseille, France),FITC-conjugated anti-CD86 (Ancell, Bayport, MN,USA), and FITC-conjugated control mouse IgG1 (Dako,Glostrup, Denmark).

PBMCs were obtained from healthy adult volunteers

Received 26 November 2013, accepted 10 December 2013Correspondence to: Toshihiro Tono, Department of Rheumatology and Infectious Diseases, Kitasato University School of Medicine1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, JapanE-mail: ttoshi@med.kitasato-u.ac.jp

118

IL-6 measurementIL-6 concentrations in the culture supernatants weremeasured using the Human IL-6 ELISA DevelopmentKit from Peprotech (Rocky Hill, NJ, USA).

RNA isolation and real-time quantitative RT-PCRTotal RNA was isolated from cultured cells using ISOGEN(Nippon Gene, Tokyo) according to the manufacturer'sspecifications. cDNA was prepared from 1μg of totalRNA using Molony murine leukemia virus (M-MLV)reverse transcriptase (Takara Bio, Otsu, Shiga) withrandom primers (Takara Bio) and was subjected to analysiswith real-time quantitative reverse transcription-polymerasechain reaction (RT-PCR) using LightCycler 4.1 (RocheDiagnostics, Lewes, UK). Real-time quantitative RT-PCRof IL-6 and β-actin was performed using SYBR Premix ExTaq II (Takara Bio) with the following primers: sense,5'-GGAGACTTGCCTGGTGAAAA-3' and antisense,5'-GTCAGGGGTGGTTATTGCAT-3' for IL-6 (genea c c e s s i o n N o . M 1 4 5 8 4 ) ; s e n s e , 5 ' -TGGCACCCAGCACAATGAA-3' and antisense, 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3' for β-actin(gene accession No. NM001101). Amplification wasperformed according to the standard protocolrecommended by the manufacturer. All results werecalibrated to the copy number of β-actin obtained fromthe same cDNA samples.

Statistical analysisStatistical significance was evaluated by Wilcoxon'ssigned rank test and paired t test where appropriate.

Results

Effects of TNF inhibitors on Annexin V expression ofSEB-stimulated monocytesInitial experiments examined the effects of TNF inhibitorson Annexin V expression of SEB-stimulated monocytesto explore their influences on the induction of apoptosis.As shown in Figure 1, TNF inhibitors, etanercept andinfliximab significantly increased the expression ofAnnexin V of monocytes compared with control IgG.These biological agents also increased the numbers ofthe Annexin V-positive and PI-negative cellssignificantly. The results, therefore, indicate that TNFinhibitors promote apoptosis of SEB-stimulatedmonocytes. Moreover, the data confirm that humanmonocytes are direct targets of TNF inhibitors.

It was previously discovered that etanercept andinfliximab were able to induce apoptosis of TNFαexpressing Jurkat T cells in the presence of human

Tono, et al.

with informed consent by centrifugation of heparinizedvenous blood over sodium diatrizorate-Ficoll gradients.Monocytes were prepared from PBMCs using theMonocyte Isolation Kit II (Miltenyi Biotec, Auburn, CA,USA). The monocyte population obtained in this mannercontained <0.1% CD3+ cells, <0.1% CD19+ cells, and>93% CD14+ cells.

The reagents, infliximab and etanercept werepurchased from Mitsubishi Tanabe Pharma (Tokyo) andTakeda Pharmaceutical (Tokyo), respectively. Thecontrol human IgG1 was purified from serum from apatient with human IgG1 myeloma using DEAE(diethylaminoethanol)-Sepharose columns. Human IgG-F(ab')2 (Gamma Venin P) was purchased from Sanofi,Paris, France.

RPMI (Roswell Park Memorial Institute) 1,640medium (Nikken, Kyoto) supplemented with penicillinG (100 U/ml) (Life technologies, Grand Island, NY,USA), streptomycin (100μg/ml) (Life Technologies),L-glutamine (0.3 mg/ml) (Sigma-Aldrich, St. Louis, MO,USA), and 10% fetal bovine serum (JRH Bio-Sciences,Lenexa, KS, USA) were used for cultures. Purifiedmonocytes (1 × 106/well) were cultured in the presenceof SEB (100 pg/ml) (Serva, Heidelberg, Germany) ineach well of 24-well flat-bottomed microtiter plates(Nunc, Roskilde, Denmark) with control IgG (10μg/ml), or pharmacologically attainable concentrations ofetanercept (10μg/ml) or infliximab (10μg/ml)8,9 for 24and 48 hours.

Analysis of cell surface antigens by flow cytometryThe surface antigens on monocytes and the induction ofapoptosis were analyzed using flow cytometry. Briefly,after a 24-hour incubation, the cells were washed oncewith a staining buffer with phosphate buffered saline(PBS) containing 2% normal human serum and 0.1%sodium azide as a preservative. The cells were thenreacted in suspension by incubating for 30 minutes at 4℃with saturating concentrations of FITC-conjugated mAbs.After the cells were washed once with a staining bufferand then twice with PBS, they were counterstained withphycoerythrin (PE)-conjugated Annexin V (R&DSystems, Minneapolis, MN, USA) according to themanufacturer's instructions. The cells were then analyzedusing Cell Lab Quanta SC (Beckman Coulter, Miami,FL, USA). To identify viable cells, gating for the stainingwith Annexin V was used. In some experiments, thecells were stained with FITC-conjugated Annexin V(R&D Systems) and propidium iodide (PI), and werethen analysed using flow cytometry.

119

Effects of TNF inhibitors on human monocytes

PBMCs, presumably through antibody dependent cellularcytotoxicity (ADCC), which requires the presence of Fcreceptors.5 It is therefore possible that interactions withFc receptors on monocytes might be required for theinduction of apoptosis of monocytes by etanercept andinfliximab. To explore this possibility, we next examinedthe influences of IgG and IgG-F(ab')2 fragments on thecapacities of these biological agents to induce apoptosisof SEB-stimulated monocytes. As shown in Figure 2,addition of IgG, but not IgG-F(ab')2 almost completelyreversed the capacities of not only etanercept, butinfliximab, to induce the apoptosis of SEB-stimulatedmonocytes (P < 0.05). These results indicate that Fcreceptors on monocytes are involved in the induction ofapoptosis of SEB-stimulated monocytes by TNFinhibitors. Moreover, it is also suggested that the outsideto inside signal via TNFα alone might not be sufficient

for the induction of the apoptosis of human monocytes.

Effects of TNF inhibitors on the expression of CD80 andCD86 on SEB-stimulated monocytesThe following experiments examined the effects of eachbiological agent on the expression of costimulatorymolecules on monocytes stimulated with SEB. Figure3A shows typical histograms of the expressions of CD80and CD86 on Annexin V-negative monocytes from anormal healthy donor. Etanercept and infliximabmarkedly suppressed the expressions of CD80 and CD86,respectively. Accordingly, both etanercept and infliximabsignificantly suppressed the expressions of CD80 andCD86 on SEB-stimulated monocytes from 9 healthyindividuals compared with the control IgG (Figure 3B).The results indicate that TNF inhibitors suppressed theexpression of costimulatory molecules on SEB-stimulated

Figure 1. Effects of TNF inhibitors on Annexin V expression of SEB-stimulated monocytes

Highly purified monocytes (1 × 106 /well) from 8 healthy individuals were cultured in the presence of SEB (100 pg/ml) in each well of24-well flat-bottomed microtiter plates with etanercept (ETN) (10μg/ml), infliximab (IFX) (10μg/ml) or control IgG (10μg/ml) for 48hours, after which the cells were stained with FITC-conjugated AnnexinV and propidium iodide (PI). The cells were then analyzed byflow cytometry. A. Representative dot plots for staining with FITC-conjugated Annexin V and PI. B. Percentages of total Annexin V-positive cells (upper) or PI-negative Annexin V-positive cells (lower) are shown. Statistical significance was evaluated by Wilcoxon'ssigned rank test.

120

monocytes.

Effects of TNF inhibitors on the IL-6 production of SEB-stimulated monocytesRecent studies have revealed that plasma concentrationof IL-6, but not TNFα, is correlated with the diseaseactivity and radiographic progression in RA.10 Theremaining experiments, therefore, examined the effectsof TNF inhibitors on the production of IL-6 of SEB-stimulated monocytes. As shown in Figure 4A, etanerceptand infliximab decreased the concentrations of IL-6 inthe culture supernatants of SEB-stimulated monocytesat pharmacologically attainable concentrations. We

subsequently examined the effects of these biologicalagents on the expression of mRNA for IL-6 in SEB-stimulated monocytes. As shown in Figure 4B, etanerceptand infliximab suppressed the expression of mRNA forIL-6 in SEB-s t imula ted monocytes a t the i rpharmacologically attainable concentrations. Theseresults indicate that etanercept and infliximab have directeffects on SEB-stimulated monocytes to suppress theirexpression of IL-6.

Discussion

This study clearly demonstrates that etanercept and

Figure 2. Differential effects of human IgG and IgG F(ab')2 fragments on the induction of apoptosis of SEB-stimulated monocytes byTNF inhibitors

Highly purified monocytes (1 × 106/well) from healthy individuals were cultured in the presence of SEB (100 pg/ml) in each well of 24-well flat-bottomed microtiter plates with etanercept (ETN) (10μg/ml), infliximab (IFX) (10μg/ml), or control IgG (10μg/ml) for 48hours, with or without IgG (100μg/ml) or IgG F(ab')2 fragments (100μg/ml). After which the cells were stained with (phycoerythrin)PE-conjugated Annexin V. The cells were then analyzed by flow cytometry. Percentages of total Annexin V-positive cells (upper, n =7) or PI-negative Annexin V-positive cells (lower, n = 2) are shown. Error bars indicate SD (standard deviation) values of 7 (upper) or2 (lower) different experiments. Statistical significance was evaluated by Wilcoxon's signed rank test.

Tono, et al.

121

Figure 3. Effects of TNF inhibitors on the expressions of CD80 and CD86 on SEB-stimulated monocytes

Highly purified monocytes (1 × 106/well) from 9 healthy individuals were cultured in the presence of SEB (100 pg/ml) in each well ofthe 24-well flat-bottomed microtiter plates with etanercept (ETN) (10μg/ml), infliximab (IFX) (10μg/ml), or control IgG (10μg/ml) for48 hours, after which the cells were stained with FITC-conjugated anti-CD80, anti-CD86, or control IgG1, followed by counterstainingwith PE-conjugated Annexin V. The cells were then analyzed by flow cytometry. A. Representative histograms of the staining of variousmolecules on Annexin V-negative monocytes. The percents positive for specific mAb staining are indicated. Stainings with isotype-matched control mAb are indicated by shade. B. Percentages positive for each specific mAb staining of monocytes from nine independentexperiments are summarized. Statistical significance was evaluated by Wilcoxon's signed rank test.

infliximab induce apoptosis of SEB-stimulatedmonocytes. Because the addition of normal human IgG,but not IgG-F(ab')2, inhibited the induction of apoptosisby these biological agents, it is suggested that Fc receptorson monocytes are involved in the induction of apoptosisof SEB-stimulated monocytes by TNF inhibitors. Aprevious study showed that ADCC is one of themechanisms of the induction of apoptosis in target cells.11

Moreover, we examined the influences of IgG-Fc on thecapacities of these biological agents to induce apoptosisof SEB-stimulated monocytes in 2 healthy individuals.The addition of IgG-Fc reversed the capacities of notonly etanercept but also of infliximab to induce apoptosisof SEB-stimulated monocytes (data not shown). Theseresults indicate that either IgG1 or IgGs has the influenceof inhibiting the induction of apoptosis by these biologicalagents. Thus, it is strongly suggested that TNF inhibitorsmight induce apoptosis of human monocytes throughADCC. It is noteworthy that Mitoma et al. demonstrated

that infliximab and adalimumab, but not etanercept,induce apoptosis of Jurkat T cells expressing TNFα onthe surface through outside to inside signals by membranebound TNFα.5 Moreover, infliximab and adalimumabexerted much higher complement dependent cytotoxicityto TNFα-expressing Jurkat T cells than did etanercept.5

It should be emphasized, however, that ADCC activitiesto TNFα-expressing Jurkat T cells of etanercept werecomparable to those of infliximab or adalimumab.5

Because the addition of intact IgG almost completelyabrogated the capacity of infliximab and etanercept toinduce apoptosis of SEB activated monocytes in thepresent study, strongly suggests that induction ofapoptosis of human monocytes by these biologicalcompounds might be mediated through ADCC.

It was found that infliximab did not result in theincreased apoptosis in the RA synovial tissue at 48 hoursafter initiation of treatment.12 However, 8 weeks oftreatment with etanercept or infliximab significantly

Effects of TNF inhibitors on human monocytes

122

increased apoptosis, accompanied by a significantdecrease in the synovial macrophage population.13

Accordingly, etanercept and infliximab induced apoptosisof macrophages and monocytes in synovial fluid in vitro.13

It should be pointed out that SEB activates monocytesand dendritic cells through the Toll-like receptor(TLR)2.14 However, previous studies demonstrated thatthe expression of TLR2 was up regulated in peripheralblood monocytes, synovial macrophages, and synovialtissue of RA, thus, playing an important role in thepathogenesis.15,16 Because TNF inhibitors inducedapoptosis of SEB-stimulated monocytes, it is possiblethat they might also induce apoptosis of monocytes invivo in RA.

Because macrophages and monocytes derived frombone marrow are precursors of type A synoviocytes,17,18

the induction of apoptosis of peripheral blood monocytesmight also lead to the reduction of synovial macrophages.These results in the present study further confirm thatone of the important mechanisms of action of biologicalagents involves the induction of apoptosis of macrophagesand monocytes in RA.

We have previously disclosed that infliximab as wellas etanercept, but not tocilizumab, inhibited theproliferation and the interferon-γ production of SEB-

stimulated T cells.7 Neither infliximab nor etanerceptinhibited the T cell activation by immobilized anti-CD3in the absence of monocytes. Because the activation of Tcells by SEB requires the presence of antigen-presentingcells,19 the inhibition of SEB-stimulated T cells byinfliximab or etanercept is considered to be a result ofthe inhibition of monocytes. Accordingly, both infliximaband etanercept suppressed the expression of HLA-DRon monocytes.7 Moreover, the data in the present studyhave shown that TNF inhibitors suppress the expressionof CD80 and CD86 on SEB-stimulated monocytes. It is,therefore, likely that the suppression of SEB-mediated Tcell activation by TNF inhibitors could be due to theinhibition of expression of HLA-DR as well as thecostimulatory molecules, CD80 and CD86. Themechanisms of suppression of expression of thesemolecules by TNF inhibitors remain unknown. Furtherstudies to elucidate the influences of TNF inhibitors onthe expression of mRNA for CD80, CD86, and HLA-DR, and on the cleavages of these molecules from themembranes, are warranted to better understand themechanisms of action.

IL-6 has been found to play an important role in thepathogenesis of RA.20,21 Successful treatment withmethotrexate22 or with TNF inhibitors23 results in the

Figure 4. Effects of TNF inhibitors on the protein expression and mRNA expression for IL-6 of SEB-stimulatedmonocytes

Highly purified monocytes (1 × 106/well) from healthy individuals were cultured in the presence of SEB (100 pg/ml)in each well of 24-well flat-bottomed microtiter plates with etanercept (ETN) (10μg/ml), infliximab (IFX) (10μg/ml), or control IgG (10μg/ml). A. After 48 hours of incubation, culture supernatants were collected and assayed forIL-6 by ELISA. Statistical significance was evaluated by Wilcoxon's signed rank test. B. After 24 hours of incubation,the cells were harvested and the expression of mRNA for IL-6 was examined by real-time quantitative RT-PCR(reverse transcription-polymerase chain reaction). Data are expressed as the ratio to copy numbers of β-actin mRNA.Statistical significance was evaluated by Wilcoxon's signed rank test.

Tono, et al.

123

reduction of plasma IL-6. However, it has also beenshown that in patients with chronic progressive neuro-Behcet's disease, who showed sustained elevation ofcerebrospinal fluid IL-6, infliximab markedly decreasedthose levels on the next day of the first infusion.24 Theresults in the current studies, together with those of thepresent study, demonstrate that TNF inhibitors suppressedthe production and the expression of mRNA for IL-6 inSEB-stimulated monocytes, confirming that TNFinhibitors inhibit the expression of IL-6 in the sites ofinflammation in vivo.

In summary, the collective results in the currentstudies, including those of this study, have delineated theinfluences of TNF inhibitors on human monocytes. Thecommon features of TNF inhibitors include the inductionof apoptosis and the inhibition of the expression of mRNAfor IL-6. The differential capacities of TNF inhibitors onT cell activation appear to be a result of their capacitiesto inhibit the expression of HLA-DR and costimulatorymolecules. Further studies are warranted to elucidate theprecise mechanisms by which TNF inhibitors regulatethe expressions of HLA-DR, CD80, and CD86.

Aknowledgments

This work was supported by Parents' Association Grantfrom Kitasato University School of Medicine and grantsfrom Mitsubishi Tanabe Pharma, Tokyo; TakedaPharmaceutical, Tokyo; and Chugai Pharmaceutical,Tokyo. These grants had no influence on the study design,the collection, analysis, or interpretation of the data, thewriting of the report, or in the decision to submit themanuscript for publication.

References

1. McInnes IB, Schett G. Cytokines in the pathogenesisof rheumatoid arthritis. Nat Rev Immunol 2007; 7:429-42.

2. Lipsky PE. Interleukin-6 and rheumatic diseases.Arthritis Res Ther 2006; 8 (Suppl 2): S4.

3. Maini R, St Clair EW, Breedveld F, et al. Infliximab(chimeric anti-tumour necrosis factor alphamonoclonal antibody) versus placebo in rheumatoidarthritis patients receiving concomitant methotrexate:a randomised phase III trial. ATTRACT StudyGroup. Lancet 1999; 354: 1932-9.

4. Fleischmann RM, Schechtman J, Bennett R, et al.Anakinra, a recombinant human interleukin-1receptor antagonist (r-metHuIL-1ra), in patients withrheumatoid arthritis: A large, international,multicenter, placebo-controlled trial. Arthritis Rheum2003; 48: 927-34.

5. Mitoma H, Horiuchi T, Tsukamoto H, et al.Mechanisms for cytotoxic effects of anti-tumornecrosis factor agents on transmembrane tumornecrosis factor alpha-expressing cells: comparisonamong infliximab, etanercept, and adalimumab.Arthritis Rheum 2008; 58: 1248-57.

6. Baldwin HM, Ito-Ihara T, Isaacs JD, et al. Tumournecrosis factor alpha blockade impairs dendritic cellsurvival and function in rheumatoid arthritis. AnnRheum Dis 2010; 69: 1200-7.

7. Tejima S, Tono T, Nagai T, et al. Differential invitro effects of biological agents on cytokineproduction of peripheral blood mononuclear cells.Kitasato Med J 2013; 43: 126-32.

8. Tracey D, Klareskog L, Sasso EH, et al. Tumornecrosis factor antagonist mechanisms of action: acomprehensive review. Pharmacol Ther 2008; 117:244-79.

9. Takeuchi T, Miyasaka N, Tatsuki Y, et al. Baselinetumour necrosis factor alpha levels predict thenecessity for dose escalation of infliximab therapyin patients with rheumatoid arthritis. Ann RheumDis 2011; 70: 1208-15.

10. Nishina N, Kaneko Y, Kameda H, et al. Reductionof plasma IL-6 but not TNF-α by methotrexate inpatients with early rheumatoid arthritis: a potentialbiomarker for radiographic progression. ClinRheumatol 2013; 32: 1661-6.

11. Curnow SJ, Glennie MJ, Stevenson GT. The role ofin antibody-dependent cellular cytotoxicity. CancerImmunol Immunother 1993; 36: 149-55.

12. Smeets TJ, Kraan MC, van Loon ME, et al. Tumornecrosis factor α blockade reduces the synovial cellinfiltrate early after initiation of treatment, butapparently not by induction of apoptosis in synovialtissue. Arthritis Rheum 2003; 48: 2155-62.

13. Catrina AI, Trollmo C, af Klint E, et al. Evidencethat anti-tumor necrosis factor therapy with bothetanercept and infliximab induces apoptosis inmacrophages, but not lymphocytes, in rheumatoidarthritis joints: extended report. Arthritis Rheum2005; 52: 61-72.

14. Mandron M, Aries MF, Boralevi F, et al. Age-relateddifferences in sensitivity of peripheral bloodmonocytes to lipopolysaccharide and Staphylococcusaureus toxin B in atopic dermatitis. J Invest Dermatol2008; 128: 882-9.

Effects of TNF inhibitors on human monocytes

124

15. Iwahashi M, Yamamura M, Aita T, et al. Expressionof Toll-like receptor 2 on CD16+ blood monocytesand synovial tissue macrophages in rheumatoidarthritis. Arthritis Rheum 2004; 50: 1457-67.

16. Radstake TR, Roelofs MF, Jenniskens YM, et al.Expression of toll-like receptors 2 and 4 in rheumatoidsynovial tissue and regulation by proinflammatorycytokines interleukin-12 and interleukin-18 viainterferon-gamma. Arthritis Rheum 2004; 50: 3856-65.

17. Burmester GR, Stuhlmüler B, Keyszer G, et al.Mononuclear phagocytes and rheumatoid synovitis.Mastermind or workhorse in arthritis? ArthritisRheum 1997; 40: 5-18.

18. Hirohata S, Yanagida T, Itoh K, et al. Acceleratedgeneration of CD14+ monocyte-lineage cells fromthe bone marrow of rheumatoid arthritis patients.Arthritis Rheum 1996; 39: 836-43.

19. Kappler J, Kotzin B, Herron L, et al. V beta-specificstimulation of human T cells by staphylococcaltoxins. Science 1989; 244: 811-3.

20. Feldmann M, Maini RN. Anti-TNF alpha therapy ofrheumatoid arthritis: what have we learned? AnnuRev Immunol 2001; 19: 163-96.

21. Wong PK, Campbell IK, Egan PJ, et al. The role ofthe interleukin-6 family of cytokines in inflammatoryarthritis and bone turnover. Arthritis Rheum 2003;48: 1177-89.

22. Frey N, Grange S, Woodworth T. Populationpharmacokinetic analysis of tocilizumab in patientswith rheumatoid arthritis. J Clin Pharmacol 2010;50: 754-66.

23. Takeuchi T, Miyasaka N, Tatsuki Y, et al. Inhibitionof plasma IL-6 in addition to maintenance of anefficacious trough level of infliximab associated withclinical remission in patients with rheumatoidarthritis: analysis of the RISING Study. Ann RheumDis 2012; 71: 1583-5.

24. Kikuchi H, Aramaki K, Hirohata S. Effect ofinfliximab in progressive neuro-Behçt's syndrome.J Neurol Sci 2008; 272: 99-105.

Tono, et al.