Inhibitory effects of an anti-rheumatic agent T-614 on immunoglobulin production by cultured B cells and rheumatoid synovial tissues engrafted into SCID mice

K. Tanaka, T. Yamamoto, Y. Aikawa, K. Kizawa, K. Muramoto1, H. Matsuno2 and A. Muraguchi3

Research Laboratories, Toyama Chemical Co., Ltd, Toyama, 1Tsukuba Research Laboratories, Eisai Co., Ltd, Ibaraki, 2Department of Orthopedic Surgery and 3 Department of Immunology, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan.

Correspondence to: K. Tanaka, Research Laboratories, Toyama Chemical Co., Ltd, Shimookui 2-4-1, Toyama 930-8508, Japan. E-mail: KEIICHI_TANAKA{at}toyama-chemical.co.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 
Objective. To clarify the pharmacological action of an anti-rheumatic agent T-614, we investigated its effects on immunoglobulin (Ig) production by cultured B cells and Ig secretion from synovial tissues of patients with rheumatoid arthritis (RA) using SCID mice engrafted with human RA tissue (SCID-HuRAg).

Methods. Murine B cells were prepared from mouse spleen by a T-cell depletion method. The cells were cultured with lipopolysaccharide (LPS) and/or interleukin 4 (IL-4) in the absence or presence of T-614. Human B cells were isolated from peripheral blood of healthy donors and the Ig production was induced by co-culture with autologous T cells and anti-CD3 antibody. SCID-HuRAg was prepared according to our previous method. T-614 was orally administered to the mice once daily for 4 weeks starting on the fourth week after the implantation. Then, peripheral blood was obtained and the implanted tissues were removed. Igs in the culture media or the sera were determined by enzyme-linked immunosorbent assay (ELISA).

Results. In murine B-cell cultures, T-614 significantly decreased not only the IgM production stimulated with LPS but IgG1 production induced by LPS and IL-4. Regarding human B cells stimulated with T cells, it also inhibited IgM and IgG production. In SCID-HuRAg mice, high concentrations of polyclonal human IgG were detectable in the sera of all mice. A significant decrease in the IgG level was observed in the T-614-treated group compared with the control group.

Conclusions. We showed that T-614 inhibited Ig production by the cultured B cells and also decreased the high level of human IgG observed in SCID-HuRAg mice. These results may support its effect on plasma Ig in RA patients and provide insights into the mechanisms of its anti-rheumatic effect.

KEY WORDS: Immunoglobulin production, T-614, Rheumatoid arthritis, Synovial tissue, SCID mouse.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 
T-614 [N-(3-formylamino-4-oxo-6-phenoxy-4H-chromen-7-yl)methanesulphonamide, Fig. 1] is a cytokine production inhibitor [13] and has been shown to display a steroid-like improvement in several autoimmune animal models, such as collagen-induced arthritis, MRL-lpr/lpr mice [4, 5] and experimental autoimmune encephalomyelitis in rats [6]. Therefore, it has attracted attention as a disease-modifying anti-rheumatic drug (DMARD) under development. In clinical trials on Japanese patients with rheumatoid arthritis (RA), T-614 has been found to yield a good therapeutic effect on the clinical symptoms and on laboratory indices, such as erythrocyte sedimentation rate (ESR), plasma C-reactive protein (CRP), levels of plasma immunoglobulin (Ig) G and IgM [7]. Now, the efficacy and tolerability of T-614 in RA patients are being compared with those of salazosulphapyridine, an established DMARD with a similar pharmacological profile, in a double-blind randomized clinical study. However, the mechanism for anti-rheumatic actions of T-614 and its immunopharmacological effect on B lymphocytes are not well understood.



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FIG. 1. Chemical structure of T-614.

 
Our present study was therefore directed to examining the effect of T-614 on Ig production as an important role of B cells. Using a model of isotype switch in murine splenic B lymphocytes and a co-culture of human B cells with anti-CD3-activated T cell, we first examined whether T-614 inhibits Ig production and the interleukin (IL) 4-induced isotype switch to IgG1. Next, we studied the effect of T-614 in a model of the severe combined immunodeficiency (SCID) mouse in which RA synovial tissue has been engrafted (SCID-HuRAg) [8, 9]. We examined whether B cells existing in synovial tissues from RA patients persistently produce Igs and whether T-614 inhibits the Ig production in this model.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 
Materials
T-614 was synthesized at Toyama Chemical Co., Ltd. (Tokyo, Japan), and used as a solution of various concentrations in dimethylsulphoxide (DMSO) or in an equimolar amount of 0.1 M NaOH. Other materials were obtained from the following sources. The culture supernatant of T24 cells was used for anti-mouse Thy-1 monoclonal antibody. Mouse anti-human CD3 monoclonal antibody (clone: X35, Cosmo Bio Co., Tokyo, Japan) was used for T-cell activation. Low-toxicity rabbit complement (Cedarlane Laboratories Ltd, Hornby, Ontario, Canada), DMEM and RPMI 1640 media (Gibco BRL/Life Technologies Inc., Rockville, MD, USA), fetal calf serum (FCS, Intergen Co., Purchase, NY, USA), lipopolysaccharide (LPS, E. coli 0127:B8, Difco Laboratories Ltd, Detroit, MI, USA), Percoll and Ficoll-PaqueTM(Amersham Pharmacia Biotech Co., Buckinghamshire, UK), recombinant mouse interleukin 4 (IL-4, Chemicon International Inc., Temecula, CA, USA), XTT (Sigma), neuraminidase (Sigma) and phenazine methosulphate (PMS, Wako Pure Chemical Co., Osaka, Japan) were obtained commercially. For enzyme-linked immunosorbent assay (ELISA) of mouse Igs, goat anti-mouse Ig antibody (ICN Pharmaceuticals Inc., Costa Mesa, CA, USA), peroxidase-conjugated rabbit anti-mouse IgG1 antibody (Zymed Laboratories, Inc., South San Francisco, CA, USA) and peroxidase-conjugated rabbit anti-mouse IgM antibody (Zymed Labs) were purchased. For ELISA of human Igs, the F(ab')2 fragment of rabbit anti-human IgM (Dako Japan, Tokyo), peroxidase-conjugated goat anti-human IgM F(ab')2 fragment (American Qualex, San Clemente, CA, USA), affinity purified rabbit anti-human IgG (EY Laboratories, San Mateo, CA, USA), and peroxidase-conjugated and affinity purified F(ab')2 fragment of goat anti-human IgG (EY Labs) were commercially available. For measurements of human IgG and IgM in serum of SCID-HuRAg mice, the following commercial kits were used: human IgG-Fc and IgM ELISA quantitation kitsTM (Bethyl Labs, Montgomery, TX, USA).

Animals
Six-week-old male BALB/c mice and 6- or 7-week-old male SCID mice (C.B-17/Icr-scidJcl) were purchased from Nippon SLC (Shizuoka, Japan) and Clea Japan (Tokyo, Japan), respectively. The animals were housed in an air-conditioned room at a temperature of 23 ± 2°C and 50 ± 20% relative humidity, under a 12 h light–dark cycle. They were fed with a commercial diet (NMF; Oriental Yeast Co., Ltd, Tokyo, Japan) and received tap water ad libitum.

Preparation of murine B cells and induction of antibody production
Murine B cells were prepared according to the method of Coffman et al. [10]. Briefly, single cell suspensions were prepared by mashing spleen fragments from three or four BALB/c mice between the frosted ends of two glass slides in FCS-free DMEM medium. To deplete T cells, the cells were incubated in the 3-fold-diluted culture supernatant of T24 cells for 30 min on ice, followed by incubation with rabbit complement at 37°C for 30 min. The remaining B cells were washed and suspended in 10% FCS–RPMI 1640 medium at a density of 4 x 106 cells/ml, and cultured for 2 days in the presence of 10 µg/ml of LPS. Then, the cells were recovered and the large activated B cells were isolated by enrichment using flotation on 50–70% Percoll gradients [11]. The cells at the 50/60% interface were collected and suspended in the medium at a density of 1 x 106 cells/ml. After the addition of T-614 (final concentrations of 0.3, 3 and 30 µg/ml), the cells were seeded at 1 x 105 cells/200 µl per well in a 96-well plate and cultured for 7 days in the presence or absence of LPS (4 µg/ml) and/or IL-4 (10 U/ml). After culture, the plate was centrifuged and 100 µl of the culture supernatant was collected and stored in a freezer (–40°C) for the determination of IgM and IgG1.

Determination of cell growth by XTT assay
Cell growth was examined using an XTT assay [12]. After the collection of medium, 50 µl of XTT solution (1 mg/ml XTT and 7.7 µg/ml PMS in RPMI 1640 medium) was added to each well and the plate was incubated for 4 h at 37°C. The absorbance at 450 nm was measured and the mitochondrial activities of the living cells were compared with those of the untreated cells.

Preparation of human B cells and induction of antibody production
Peripheral blood mononuclear cells were obtained from healthy volunteers by centrifugation of heparinized venous blood over Ficoll-Paque. The cells were washed twice with medium and then incubated with neuraminidase-treated sheep red blood cells (N-SRBC) for 2 h at 4°C. The rosetting and non-rosetting populations were then separated by centrifugation on Ficoll-Paque. The non-rosetting cells at the interface were collected as the B-cell population and washed with 10% FCS–RPMI 1640 medium. The T-cell population of sedimented N-SRBC rosette-forming cells was treated with isotonic NH4Cl to lyse N-SRBC and suspended in the medium. Cultures were carried out in triplicate in a total volume of 200 µl. After the addition of T-614 (final concentrations of 0.03–10 µg/ml), B cells (1 x 105 cells/well) and the autologous T cells (2 x 105 cells/well) were seeded in a 96-well plate and cultured for 6 days in the presence or absence of anti-human CD3 (1 µg/ml). Then, the culture supernatant was recovered for the determination of IgM and IgG.

Determination of Igs in culture supernatant by ELISA
A modification of the methods of Coffman and Carty [13] was employed for isotype-specific ELISA. Briefly, the concentrations of mouse IgG1 and IgM, and human IgM and IgG in culture supernatants were determined by sandwich ELISAs with the following reagents. Peroxidase-conjugated anti-mouse IgG1 or anti-mouse IgM rabbit antibody was used to assess the quantity of Igs bound to the goat anti-mouse Ig antibody-coated wells. Peroxidase-conjugated anti-human IgM or anti-human IgG goat antibody was used to assess the quantity of Igs bound to the rabbit anti-human Ig antibody-coated wells. For standard curves, mouse IgG1 kappa and IgM lambda 1 (ICN Pharmaceuticals) or human IgM (Rockland Immunochemicals, Gilbertsville, PA, USA) and IgG (Zymed Labs) were used in the corresponding ELISAs.

Preparation of SCID-HuRAg mice and treatment with T-614
SCID-HuRAg mice were prepared according to our previous reports [8, 9, 1416]. Fresh rheumatoid synovium was obtained after informed consent at the time of joint surgery from four RA patients. The excised synovial tissues were used for implants and the size of the specimen was adjusted to a block of 5–10 mm in diameter. SCID mice were anaesthetized with diethyl ether and the tissue implants were grafted subcutaneously into the backs of the mice. All surgical procedures were performed under aseptic conditions.

A total of 16 SCID-HuRAg mice were used in this study. The mice were randomly assigned to two groups at 4 weeks after the implantation. One group of mice was orally administered T-614 at a dose of 100 mg/kg once daily for 4 weeks and the other was given sterile distilled water as a control. A normal group consisting of six SCID mice with no implants were left untreated during the experimental period. On the day after the final administration, the mice were anaesthetized and their blood was collected.

Histochemical analysis
The implanted tissues were removed together with murine subcutaneous tissues and histologically observed following haematoxylin and eosin staining. In order to examine biological activities of the implanted tissues, immunohistochemical staining was performed as previously described [8, 9]. The sections were stained with the following anti-human monoclonal antibodies and an immunostaining kit: CD20 (L26), HLA-DP,DQ,DR (CR3/43) and EnVision+TM. These were purchased from Dako Japan.

Measurement of human IgG and IgM in the serum of SCID mice
Serum concentrations of human polyclonal IgG and IgM were measured using the above-mentioned commercial kits according to manufacturer's instructions. The sera from non-implanted SCID mice and normal ICR mice were used as negative controls.

Statistical analysis
All results are expressed as the mean ±standard error of the mean (S.E.M.). Statistical comparisons among the groups in culture experiments were carried out by Dunnett's multiple comparison following a randomised block design (SAS release 6.12; SAS Institute Inc., Cary, NC, USA). For serum parameters in SCID-HuRAg mice, Wilcoxon rank sums test was used in the comparison between two groups. The inhibition percentage was calculated from the ratio to each corresponding control. IC50 values were calculated using a logistic regression analysis (SAS release 6.12).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 
Inhibition of Ig production and the isotype switch in murine B cells
To examine the effect of T-614 on maturation of B cells to plasma cells, murine B cells were incubated with LPS and/or IL-4, and the resultant levels of IgM and IgG1 in culture supernatants were measured. In non-stimulated and IL-4-stimulated conditions, T-614 did not affect, or only marginally affected, IgM production. However, at 3 and 30 µg/ml, T-614 significantly inhibited the elevation induced by the addition of 4 µg/ml LPS. The IgM secretion induced by the simultaneous addition of LPS and IL-4 was also decreased to a non-stimulated level by T-614 (Fig. 2 left). In addition, T-614 inhibited IgG1 production stimulated with the simultaneous addition of LPS and IL-4. The inhibition rates at 3 and 30 µg/ml of T-614 were 73 and >90%, respectively (Fig. 2 right).



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FIG. 2. Effect of T-614 on IgM and IgG1 production in murine splenic B cells. The cells were cultured in the presence or absence of LPS (4 µg/ml) and/or IL-4 (10 U/ml). T-614 was added to the cultures at concentrations of 0 (control, white column), 0.3 (light grey column), 3 (dark grey column) and 30 (black column) µg/ml. After 7 days, culture supernatants were collected, and secreted IgM and IgG1 levels were measured by ELISAs. Data are shown as the mean ± S.E.M. of three experiments. *P < 0.05 compared with corresponding control by Dunnett's multiple comparison following a randomized block design.

 
Effect on B-cell proliferation
The proliferation assay by XTT revealed that both LPS and IL-4 showed an increasing effect on cell numbers, and in the simultaneous addition there was a 28-fold increase compared with non-stimulated cultures. T-614 at 30 µg/ml inhibited the LPS/IL-4-induced proliferation by about 50%; however, at 3 µg/ml, which showed a significant inhibition of Ig production, it did not affect the cell growth induced by LPS, IL-4 or both (Fig. 3).



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FIG. 3. Effect of T-614 on cell proliferation in murine splenic B cells. The cells were cultured in the presence or absence of LPS (4 µg/ml) and/or IL-4 (10 U/ml). T-614 was added to the cultures at concentrations of 0 (control, white column), 0.3 (light grey column), 3 (dark grey column) and 30 (black column) µg/ml. After 7 days culture, cell viability was measured by the XTT method. Data are shown as the mean ± S.E.M. of three experiments. *P < 0.05 compared with corresponding control by Dunnett's multiple comparison following a randomized block design.

 
Inhibition of Ig production in human B cells
To examine the effect of various concentrations of T-614 on Ig production by human B cells, peripheral B cells were stimulated by a 6-day co-culture with autologous T cells and anti-CD3 antibody, and Ig levels were measured, though IgG could not be assessed separately by specific isotypes. As shown in Fig. 4, T-614 suppressed the production of IgM and IgG in a dose-dependent manner with IC50 values (n = 3) of 0.58 ± 0.26 and 0.55 ± 0.12 µg/ml, respectively.



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FIG. 4. Effect of T-614 on IgM and IgG production by human B cells stimulated with anti-CD3- activated T cells. B cells from three healthy donors were stimulated by a 6-day co-culture with autologous T cells and anti-CD3 antibody in the presence or absence of T-614 (0.03–10 µg/ml). After 6 days, culture supernatants were collected, and secreted IgM (A) and IgG (B) levels were measured by ELISAs. Data are shown as the mean ± S.E.M. of three donors.

 
Effect on SCID-HuRAg mice
In SCID-HuRAg mice, there was no abnormal reaction in either the control or T-614-treated groups during the entire experimental period. No statistically significant difference in body weight gains was noted: 0.2 ± 0.4 g (n = 8) in the control group, 0.7 ± 0.2 g (n = 8) in the T-614 group and 1.3 ± 0.2 g (n = 6) in the normal group. Thus, T-614 at a daily dose of 100 mg/kg exhibited no toxic effect on SCID-HuRAg mice.

Although neither human IgG nor IgM was detected in sera of normal SCID mice and ICR mice (IgG <0.11 µg/ml; IgM <0.53 µg/ml), a high level (84.4–2265 µg/ml) of human IgG and a low level (1.32 ± 0.24 µg/ml) of human IgM were observed in control SCID-HuRAg mice (Fig. 5). There was a considerable variation in IgG levels between the grafts from different patients: some mice with an identical graft showed high levels of 2265 µg/ml in the control group and 479 µg/ml in the T-614-treated group, while other mice with a different graft showed low levels, 84 µg/ml and 36 µg/ml, respectively. In the T-614-treated SCID-HuRAg group, regardless of such dispersion, there was a significant decrease of the IgG level and the mean value was about one-half of that in the control group. On the contrary, there was no significant difference in IgM level between the two groups.



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FIG. 5. Effect of T-614 on human IgG and IgM levels in serum of SCID-HuRAg mice, an RA model using synovial tissue transplanted into SCID mice. Mice in the normal group (n = 6) were neither implanted nor treated. SCID-HuRAg mice were divided into two groups (n = 8) 4 weeks after the transplantation and T-614 or vehicle (control) was orally administered once daily for 4 weeks. After 4 weeks, serum samples were obtained from each mouse, and the human IgG (A) and IgM (B) levels were measured by ELISAs. Each circle represents the measured value of an individual mouse and the horizontal bar with vertical line represents the mean with S.E.M. of eight mice. *P < 0.05 compared with control by Wilcoxon rank sums test. NS, not significant.

 
Figure 6 shows histological features of the engrafted synovial tissue from a control SCID-HuRAg mouse 8 weeks after implantation. Pathological conditions such as stratification of synovial cells, infiltration of inflammatory cells and formation of lymphoid follicles were observed in a large part of the sections (Fig. 6A). In addition, there was an infiltration of numerous plasma cells that could be histologically discriminated (Fig. 6B) and CD20-positive cells in the centre of the follicle (Fig. 6D). Immunostaining with anti-HLA-DP,DQ,DR antibody made clear the boundary layer between human synovium and murine subcutaneous tissues, in which most of the synovial fibroblasts and infiltrated cells were positive (Fig. 6C). These observations were similar to our previous reports [9, 14, 16].



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FIG. 6. Light microscopic features of implanted tissue in SCID-HuRAg mice in the control group. (A) Haematoxylin and eosin staining (original magnification x25); (B) typical plasma cells were observed (arrows) (original magnification x100); (C) immunohistochemical staining with anti-human HLA-DP,DQ,DR antibody; and (D) with anti-human CD20 antibody (original magnification x25).

 
Examination of the T-614-treated group showed that there was no apparent difference in histological features of the implanted tissue from those of the control group. The number of inflammatory cells (407 ± 36 cells/mm2, n = 8) detected in this group was almost comparable with that (378 ± 32 cells/mm2, n = 8) in the control group. The number of infiltrating plasma cells in the treated group was also similar to that in the control group (data not shown).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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 References
 
Mature B cells expressing IgM are capable of switching to any antibody class during the differentiation process to plasma cells, resulting in the secretion of IgG, IgE or IgA in place of IgM [17]. It has been known that cytokines produced by helper T cells are involved in this isotype switch; IL-4 drives the switch to IgG1 or IgE, and interferon-{gamma} induces the switch to IgG2a [18]. In the present study using murine splenic B cells, the production level of IgM was not affected by IL-4, but that of IgG1 was elevated by the simultaneous addition of LPS and IL-4, showing that switch to IgG1 from IgM was induced by IL-4 as demonstrated previously [18] (Fig. 2).

T-614, at concentrations of 3 µg/ml or more, inhibited both IgM and IgG1 production by the B cells (Fig. 2). Since such an effect was observed in the T-cell-depleted culture, it suggests that the inhibition of Ig production by T-614 is due to its direct effect on B cells. Furthermore, in human B cells stimulated with anti-CD3-activated T cells, T-614 also suppressed IgM and IgG production at a lower concentration (Fig. 4). Of note is that the inhibition of IgG1 production by T-614 was more effective than that of IgM, indicating that T-614 may affect the process of class switch induced by IL-4. Since T-614 at 3 µg/ml had no effect on B-cell proliferation (Fig. 3), it is unlikely that its cellular toxicity or the suppression of proliferation was related to the effect on Ig production. Thus, for the inhibition of Ig production by T-614, a different mechanism from the suppression of proliferation might be involved. Although the mechanism of T-614 for the effect on B-cell function has not been clarified, this study suggests that T-614 may have an inhibitory effect on IL-4 signal transduction. Further studies will be required to investigate the effect of T-614 on cellular signal transduction in the IL-4-induced class switch mechanism in B cells.

Using a SCID-HuRAg mouse model, we examined whether B cells existing in synovial tissues from RA patients persistently produce Igs and whether T-614 inhibits Ig production in this model. In the present study, a high level of human polyclonal IgG was detected in serum of all the mice after the implantation. Human IgM was also found, but the level was much lower (Fig. 5). The high levels of human IgG in mouse serum must derive from CD20-positive B cells and histologically discriminated plasma cells in the implant tissues (Fig. 6).

T-614 at a daily dose of 100 mg/kg showed a significant inhibition of human IgG produced by the implanted tissues (Fig. 5). This is the first experimental evidence that supports the clinical improvement effect of DMARDs on plasma Ig levels of RA patients. The dose of T-614 inhibiting the IgG production exceeded the doses (50 or 75 mg/day) yielding clinical efficacy. However, pharmacokinetics of T-614 were found to have species differences and the data revealed that the area under the curve (AUC) in mice was about 20-fold lower than that in humans when given an identical dose (data not shown). In fact, we reported that its effective doses in mouse arthritis models, such as collagen-induced arthritis and MRL/lpr mice, were 10 mg/kg or more [4]. Therefore, the dose used in this study is considered to be reasonable, and sufficient efficacy of T-614 on Ig production by synovial tissue may be expected in clinical studies. T-614 did not induce apparent histological changes in RA synovial tissues in this model; how-ever, we have already reported that among well-recognized DMARDs including salazosulphapyridine, only methotrexate inhibits synovitis by inducing apoptosis [14].

Matsumoto et al. [19] have reported a mouse model for RA in which the pathology is driven almost entirely by the Ig recognizing a ubiquitously expressed protein, glucose-6-phosphate isomerase (GPI). They have proposed that some forms of RA may develop by a mechanism fundamentally different from the currently approved paradigm of joint-specific T-cell response, i.e. a revival of the B-cell paradigm for RA pathogenesis [20]. Recently, it has also been found that 64% of humans with RA had increased concentrations of anti-GPI IgG in serum [21]. Moreover, Edwards and Cambridge [22] have shown a clinical efficacy of B-lymphocyte depletion by a monoclonal anti-CD20 antibody in RA patients and it has been suggested that RA is critically dependent on B lymphocytes. Thus, the inhibition of Ig production by T-614 may be directly involved in its clinical efficacy in RA, although this is only speculation.

In conclusion, T-614 inhibited IgM production and the isotype switch to IgG1 induced by IL-4 via a direct effect on B cells. Furthermore, IgM and IgG production by human B cells was also inhibited in a dose-dependent manner. In SCID-HuRAg mice, human IgG was detected in the serum at a high concentration and T-614 treatment resulted in a significant suppression of the IgG accumulation in the mouse serum. These results may support its improvement effect on plasma Igs in clinical trials on RA patients and provide insights into the mechanisms for its anti-rheumatic effect.


    Conflict of interest
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 
The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conflict of interest
 References
 

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Submitted 20 August 2002; Accepted 19 March 2003





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