Effects of a pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L. on interleukin 6 production of murine B cells and B cell lines

Immunopharmacology 49 Ž2000. 307–316 www.elsevier.comrlocaterimmpharm

Effects of a pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L. on interleukin 6 production of murine B cells and B cell lines Yingjie Guo a,c , Tsukasa Matsumoto a , Yuji Kikuchi b, Takashi Ikejima c , Benxiang Wang c , Haruki Yamada a,b,) a

Oriental Medicine Research Center, The Kitasato Institute, 5-9-1, Shirokane, Minato-ku, Tokyo 108-8642, Japan b Center for Basic Research, The Kitasato Institute, 5-9-1, Shirokane, Minato-ku, Tokyo 108-8642, Japan c Research Center of New Drug, Changchun College of Traditional Chinese Medicine, Chang Chun, Jilin 130021, People’s Republic of China Accepted 2 May 2000

Abstract The effect of T-cell-independent B cell mitogen, a pectic polysaccharide, bupleuran 2IIc, from a medicinal herb, the roots of Bupleurum falcatum L. on interleukin 6 ŽIL-6. production of murine B cells and B cell lines was investigated in order to clarify the mechanism of enhanced immunoglobulin ŽIg. secretion from B cells. Bupleuran 2IIc enhanced IgM secretion from highly purified murine normal B cells. When normal B cells from murine spleen were cultured with bupleuran 2IIc in the presence of anti-IL-6 neutralizing antibody, the enhanced IgM secretion by bupleuran 2IIc was reduced. When B cells were stimulated with bupleuran 2IIc, their IL-6 secretion and the transcription of IL-6 mRNA were enhanced. The enhanced IL-6 secretion by bupleuran 2IIc was also observed in B cell line, Y16 cell. When bupleuran 2IIc was digested with endo-a-Ž1 4.-D-polygalacturonase, the resulting enzyme resistant carbohydrate portion, AramifiedB region ŽPG-1. composed of rhamnogalacturonan core containing neutral sugar side chains showed potent IL-6 secretion-enhancing activity. These results suggest that the AramifiedB region of bupleuran 2IIc stimulates the secretion of IL-6 as the active site, and the resulting IL-6 may partially contribute the enhancement of IgM secretion as an autocrine andror paracrine mechanism. q 2000 Elsevier Science B.V. All rights reserved.

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Keywords: Bupleurum falcatum; Polysaccharide; B cell; Interleukin 6

1. Introduction

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Corresponding author. Oriental Medicine Research Center, The Kitasato Institute, 5-9-1, Shirokane, Minato-ku, Tokyo 1088642, Japan. Tel.: q81-3-3444-6161; fax: q81-3-3445-1351. E-mail address: [email protected] ŽH. Yamada..

The roots of Bupleurum falcatum L. have been widely used for the treatment of chronic hepatitis, kidney syndrome, inflammatory diseases and ulcer of digestive organs in Chinese and Japanese herbal medicines. The roots of B. falcatum contain several

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saikosaponins ŽAimi et al., 1968., and their various pharmacological activities have been reported ŽTakagi and Shibata, 1969; Abe et al., 1982, 1986; Mizoguchi et al., 1985.. However, the efficacy of B. falcatum L. cannot be explained by the action of saponin alone. The pharmacologically active polysaccharides, bupleuran 2IIb and 2IIc have been isolated from the hot water extract of the root of B. falcatum ŽYamada et al., 1989. and anti-complementary ŽYamada et al., 1989., macrophages Fc receptor up-regulating ŽMatsumoto et al., 1993. and anti-ulcer activities ŽSun et al., 1991; Yamada et al., 1991a. have been observed as their pharmacological activities. Bupleuran 2IIc was characterized as a pectin-like polysaccharide consisting of galacturonan region, AramifiedB region Žwhich is composed of the rhamnogalacturonan core with neutral sugar side chains. and rhamnogalacturonan II-like region ŽYamada et al., 1991b; Hirano et al., 1994., and the AramifiedB region has been considered as an important part for the expression of these activities ŽYamada et al., 1989; Sakurai et al., 1999.. Recently, we demonstrated that bupleuran 2IIc also induced the proliferation and maturation of murine B lymphocytes in vitro and in vivo, and increased nonspecific immunoglobulin ŽIg. secretion from the B cells ŽSakurai et al., 1999.. This secretion was more enhanced in coexistence with interleukin 6 ŽIL-6. than in the case of just bupleuran 2IIc alone ŽSakurai et al., 1999.. IL-6 is a pleiotropic cytokine influencing the antigen-specific immune response ŽTakatsuki et al., 1988. and inflammatory reaction ŽHirano, 1992.. Because IL-6 is one of the major factors for the terminal differentiation of activated B cells into Igsecreting cells ŽMuraguchi et al., 1988., it assumes a possibility that IL-6 may involve in Ig secretion-enhancing activity of B cells by bupleuran 2IIc. From an immunopharmacological study of bupleuran 2IIc on B cells, we now found that the polysaccharide enhances the IL-6 secretion from B cells, although such enhancement by plant polysaccharides on IL-6 secretion from B cells has not yet been reported. In the present paper, we studied the effects of bupleuran 2IIc on IL-6 production of murine B cells and B cell lines in order to know the mechanism of Ig secretion-enhancing activity of bupleuran 2IIc.

2. Materials and methods 2.1. Animals Specific pathogen-free C3HrHeJ and C3HrHeN female mice Ž6–8 weeks old. were obtained from SLC ŽShizuoka, Japan.. They were given free access to standard laboratory chow ŽCE-2, CLEA Japan. and water. 2.2. Reagents RPMI 1640 medium, Hanks’ balanced salt solution ŽHBSS., phosphate-buffered saline ŽPBS. were purchased from Gibco ŽGrand Island, NY, USA.. Fetal bovine serum ŽFBS. was purchased from Cell Culture Laboratories ŽCleveland, OH, USA.. Penicillin, streptomycin, and amphotericin B were from Flow Laboratories ŽIrvine, Scotland.. Alamar Bluee was purchased from Alamar Bio-Sciences ŽSacramento, CA, USA.. B cell lines, SP2 Žmyeloma line; Shulman et al., 1978., Y16 ŽIL-5 dependent early B cell line; Takaki et al., 1990., and BCL1-B20 Žmurine B cell chronic leukemic line; Mita et al., 1988. were kindly provided by Dr. K. Takatsu, Institute of Medical Science, University of Tokyo, and IL-6 dependent murine hybridoma clone, MH-60 ŽMatsuda et al., 1988. was obtained from Dr. M. Hayashi, Center for Basic Research, The Kitasato Institute. 2.3. Preparation of bupleuran 2IIc and subfractions The roots of B. falcatum L., whose quality is under the control of the Japanese Pharmacopoeia Ž13th edn.., were purchased from Uchida Wakanyaku ŽTokyo, Japan.. A voucher specimen was deposited at the herbarium of Oriental Medicine Research Center of the Kitasato Institute. Bupleuran 2IIc was purified from pectic polysaccharide fraction ŽBR-2. of the roots of B. falcatum by anion-exchange chromatography on DEAE-Sepharose CL-6B ŽPharmacia Fine Chemicals, Uppsala, Sweden. as described previously ŽSun et al., 1991.. ARamifiedB region, ŽPG-1; rhamnogalacturonan core possessing side chains rich in neutral sugars., PG-2 Žrhamnogalacturonan II-like region. and PG-3 Žoligogalacturonide fraction. were prepared from bupleuran 2IIc by endo-Ž1 4.-a-Dpolygalacturonase digestion followed by gel filtration as reported previously ŽSun et al., 1991..

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2.4. Preparation and purification of B cells from spleen The mice were sacrificed by cervical amputation, and spleens were separated by removing the adhering fat and connective tissue using aseptic techniques. Spleen cells were dispersed by pressing with the flat surface of a syringe plunger against the sterilized stainless sieve Ž150 mesh. on the tissue culture dish. The spleen cell suspensions were filtered through a 200-mesh sterilized stainless sieve and washed twice with HBSS supplemented with 1% BSA ŽHBSS–BSA.. B cells were purified from spleen cell suspension by magnetic cell sorting ŽMACS. system. Briefly, after passing through the 40-mm nylon mesh, the spleen cells were incubated for 20 min at 48C with anti-mouse CD45RrB220 MicroBeads ŽMiltenyi Biotec, Bergisch Gladbach, Germany. at a density of 100 ml antibody solutionr10 8 spleen cells. The labelled cells were washed once with PBS containing 2 mM EDTA and 0.5% BSA ŽPBS–EDTA–BSA. and applied to a MACS separation column Žtype VSq, Miltenyi Biotec. within the magnetic field. A non-magnetic cell fraction was eluted from the column with 3–5 column volumes of degassed PBS–EDTA–BSA. The labelled cells were eluted with degassed PBS– EDTA–BSA after the column had been demagnetized by removal from the magnetic field. In order to increase the purity of B cells, the separated positive cell fraction was reapplied to a new MACS separation column and repeated the column separation. The cell viability was uniformly greater than 98% as determined by Trypan blue exclusion, and always more than 99% of the cells showed lymphocyte morphology with characteristic staining. The efficiency of the separation was determined by flow cytometry, and the cells obtained were over 98% CD45RrB220 and CD19 double positive B cells Ždata not shown.. 2.5. Alamar Bluee reduction assay Cell growth was measured by a fluorometric assay, Alamar Bluee reduction assay ŽPage´ et al., 1993.. Cells Ž100 ml. were incubated in 96-well plates for 3 days in a humidified atmosphere of 5% CO 2 –95% air. At 4 h prior to culture termination, 10

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ml of Alamar Bluee solution was added to each well, and the cells were then continuously cultured. The fluorescence intensity was measured by Fluoroskan II ŽLabsystems Oy, Helsinki, Finland. at an excitation wavelength of 544 nm and emission wavelength of 590 nm. The delta soft II ŽVer 4.13 FL, BioMetallics, Princeton, NJ. was used for data management. 2.6. Measurement of IgM by ELISA The microtiter plate for ELISA ŽMaxiSorp a F96, Nunc, Roskide, Denmark. was coated overnight at 48C with 1 mgrml of purified goat anti-mouse Ig ŽIgA, IgM and IgG, Capel, USA. in 50 mM bicarbonate buffer ŽpH 9.6.. After washing four times with PBS containing 0.05% Tween 20 ŽPBS-Tween., the plate was blocked with 0.8% gelatin in PBS at 378C for 1 h. The harvested supernatants of B cells were added to the first antibody-coated wells in 100 ml aliquots and the plate was incubated at 378C for 1 h. After the plate was washed four times with PBSTween, 100 ml of 1r1000 dilution of alkaline phosphatase labelled goat anti-mouse IgM ŽOrganon Teknika, USA. in PBS-Tween was added to each well and the plate was incubated at 378C for 1 h. After washing the wells with PBS-Tween four times, each well was incubated with 150 ml of chromogenic substrate solution Ž1 mg of p-nitrophenylphosphate disodium salt in 1 ml of 10% diethanolamine buffer, pH 9.8., and subsequently the absorbance at 405 nm was measured using a microplate reader ŽBio-Rad, Model 250, Nippon Bio-Rad, Tokyo, Japan.. 2.7. Determination of IL-6 contents 2.7.1. ELISA for IL-6 A solution Ž2 mgrwell. of purified anti-IL-6 mAb Žclone MP5-20F3, PharMingen, San Diego, CA, USA. in 50 ml of PBS was added to microtiter plate ŽMaxiSorpe F96. for overnight at 48C. After four times washing with PBS-Tween, the plate was further incubated with 1% BSA in PBS at room temperature for 2 h Žblocking.. Samples were added to the first antibody-coated well at 100 mlrwell, and the plates were incubated at room temperature for 1 h. For the standard curve, recombinant murine IL-6 ŽGenzyme, Cambridge, MA, USA. was used. After

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the plate was washed four times with PBS-Tween, 100 ml of biotinylated anti-mouse IL-6 mAb ŽMP532C11, PharMingen. in PBS containing 10% fetal calf serum was added to the wells, and the plate was incubated for 4 h at room temperature. Then the plate was washed six times with PBS-Tween. Alkaline phosphatase labelled streptavidin ŽGibco, Grand Island, NY, USA. diluted with PBS-Tween containing 1% BSA Ž1:2000., was added to each well and the plates were then incubated at room temperature for 1 h. After washing the wells with PBS-Tween eight times, each well was incubated with 150 ml of chromogenic substrate solution Ž1 mg of pnitrophenylphosphate disodium salt in 1 ml of 10% diethanolamine buffer, pH 9.8., and the absorbance was measured as described above. Detection limit of ELISA for IL-6 is 42 pgrml Ždata not shown.. 2.7.2. Determination of bioactiÕe IL-6 IL-6 activity was detected by bioassay of IL-6 dependent murine hybridoma clone, MH-60 ŽMatsuda et al., 1988.. MH-60 cells were washed three times with RPMI 1640–10% FBS, and seeded in microplates at a density of 5 = 10 3 cellsrwell in RPMI1640–15% FBS. One hundred microliters of serial dilution of standard IL-6 or harvested supernatant of B cells and 50 ml of RPMI 1640–10% FBS were added to each well, respectively. The final volume is 200 mlrwell. After a 72-h incubation Ž378C, humidified 5% CO 2 ., cell growth was measured using Alamar Bluee reduction assay as described above. 2.8. RNA preparation and polymerase chain reaction (PCR) The B cells were lysed by ISOGEN w ŽNippon Gene, Tokyo, Japan., and the preparation of total RNA from the lysate was performed according to the supplier’s instruction manuals ŽNippon Gene.. cDNA was prepared from total RNA Ž2 mg. by Maloney murine leukemia virus ŽMMLV. reverse transcriptase using Ready To Goe T-primed FirstStrand Synthesis Kit according to the supplier’s instructions ŽPharmacia Biotech, Uppsala, Sweden., and frozen at y208C until use. The resulting cDNA was subjected to PCR as follows. Briefly, 5 ml cDNA solution was added to 45 ml of PCR mixture

containing 11.1 mM Tris–HCl ŽpH 8.3., 55.5 mM KCl, 1.56 mM MgCl 2 , 0.22 mM deoxynucleoside triphosphates, 1.11 mM of specific primers, and 5 units of recombinant Taq DNA polymerase ŽTaKaRa, Tokyo, Japan.. Each 50-ml sample was amplified in a HotStart 50e Storage and Reaction Tube ŽMolecular Bio-Products, San Diego, CA. using DNA thermal cycler, type PC-700 ŽASTEC, Fukuoka, Japan.. An initial PCR cycle at 948C for 2 min was carried out, followed by 35 cycles of PCR using the following temperature profile: denaturation at 948C for 0.5 min, 1 min at 608C for annealing, 1 min at 728C for extension. The extension reaction of final cycle was performed for 10 min at 728C. A sense primer and an antisense primer were synthesized according to published cDNA sequences for murine IL-6 ŽChiu et al., 1988. and cyclophilin ŽHasel and Sutcliffe, 1990., the products sequences were defining a 229-base for IL-6, and 372-base for cyclophilin target sequences, respectively. The actual sequences of the oligonucleotides were as follows: IL-6 sense 5X-GAC AAA GCC AGA GTC CTT CAG AGA G-3X , antisense 5X-CTA GGT TTG CCG AGT AGA TCT C-3X , cyclophilin sense 5X-GGT CAA CCC CAC CGT GTT CTT CGA-3X , antisense 5X-TTG CCA TCC AGC CAT TCA GTC TTG-3X . The oligonucleotides for primer were synthesized on a Model 394 DNA synthesizer ŽApplied Biosystems.. The cyclophilin primers were used as a control for reverse transcriptase reaction and PCR. All amplified cDNA were analyzed by 1.5% agarose gel electrophoresis and stained with ethidium-bromide for visualization under UV illumination, and photographed using Polaroid film, type 667 ŽPolaroid.. The size of amplified DNA was identified by comparison with DNA marker Ž100 bp DNA ladder, New England Biolabs, Beverly, MA.. PCR-assisted amplification was repeated twice, and similar results were obtained Ždata not shown.. 2.9. Statistics Values are expressed as mean " S.D. The differences between the control and the treatment in these experiments were tested for statistical significance by Student’s t-test. A value of p - 0.05 was considered to indicate statistical significance.

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3. Results 3.1. Effect of bupleuran 2IIc on IgM secretion of purified B cells Previous observations have demonstrated that bupleuran 2IIc enhances IgG and IgM secretions from whole spleen cells ŽSakurai et al., 1999.. In order to know whether this enhancement of Ig secretion by bupleuran 2IIc is T cell- andror macrophage-dependent or not, we assessed the IgM secretion-enhancing activity of bupleuran 2IIc using highly purified murine spleen B cells. As shown in Fig. 1, bupleuran 2IIc increased the IgM secretion of purified B cells from both LPS-non-responsive C3HrHeJ mouse and

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LPS-responsive C3HrHeN mouse to the same extent. These results indicate that the enhancement of IgM secretion by bupleuran 2IIc is dependent on B cell but not on T cell and macrophage. The results also suggest that enhanced IgM production is not due to the contamination of LPS. 3.2. Effect of anti-IL-6 antibody on enhancement of IgM secretion from B cells by bupleuran 2IIc Because IL-6 is one of the major factors for the terminal differentiation of activated B cells into Igsecreting cells and enhances Ig secretion ŽMuraguchi et al., 1988., it is a presumed possibility that IL-6 may be involved in the Ig secretion-enhancing activity of B cells by bupleuran 2IIc. To determine whether IL-6 is involved in the enhancement of Ig secretion from normal B cells by bupleuran 2IIc, the effect of anti-IL-6 neutralizing antibody on the enhancement of IgM secretion from murine spleen B cells by bupleuran 2IIc was examined. The presence of anti-IL-6 neutralizing antibody alone did not affect the spontaneous secretion of IgM from B cells ŽFig. 2.. However, the enhancement of IgM secretion by bupleuran 2IIc was reduced in the presence of anti-IL-6 neutralizing antibody ŽFig. 2.. The enhancement of IgM secretion by bupleuran 2IIc was reduced almost completely by 48 h, and partially reduced by 72 h ŽFig. 2.. These results suggest that IL-6 contributes to the enhancement of IgM secretion from B cells by bupleuran 2IIc. 3.3. Effects of bupleuran 2IIc on the IL-6 production of B cells

Fig. 1. Effect of bupleuran 2IIc on IgM secretion from normal B cells. Purified B cells Ž2=10 6 cellsrml. from ŽA. C3HrHeJ mouse or ŽB. C3HrHeN mouse were cultured with or without bupleuran 2IIc Ž0.1 mgrml. until the designated time. IgM in the culture supernatants was measured by ELISA. `: control, v: bupleuran 2IIc. Data were expressed as mean"S.D. Ž ns 4..

B cells are known to be able to secrete IL-6 ŽHirano et al., 1990.. In order to know whether bupleuran 2IIc induces IL-6 secretion from B cells, purified B cells from murine spleen were cultured in the presence of bupleuran 2IIc and then the conditioned medium was subjected to ELISA and bioassay for IL-6. The ELISA result showed that the IL-6 content in the conditioned medium was increased significantly in a time-dependent manner compared with control ŽFig. 3A.. Through the bioassay using MH-60 cell, the bioactive IL-6 was upregulated from 48 to 96 h ŽFig. 3B..

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scription of B cells was analyzed by RT-PCR. When B cells were stimulated with bupleuran 2IIc for 72 h, the enhanced transcription of IL-6 mRNA was detected in activated B cells ŽFig. 4.. The results indicate that IL-6 is newly produced and secreted from B cells by the stimulation of bupleuran 2IIc. 3.4. IL-6 production from bupleuran 2IIc-treated B cell line Next, we examined whether enhanced IL-6 secretion is also induced by bupleuran 2IIc treatment of B cell line. Three B cell lines — SP2, BCL 1-B20 and Y16, which were characterized and classified as myeloma, immature B and early B cells, respectively — were used for the assay. The cultured super-

Fig. 2. Effect of anti-IL-6 Ab on enhancement of IgM production from bupleuran 2IIc-treated normal B cells. Purified B cells Ž2=10 6 cellsrml. from C3HrHeJ mice were cultured with bupleuran 2IIc Ž0.1 mgrml., anti-IL-6 Ab Ž20 mgrml., anti-IL-6 Ab Ž20 mgrml.qbupleuran 2IIc Ž0.1 mgrml. or culture medium for 48 or 72 h. IgM in the culture supernatants was measured by ELISA. Data were expressed as mean"S.D. Ž ns 4..

Bupleuran 2IIc shows potent mitogenic activity on B cells ŽSakurai et al., 1999.. Therefore, in order to exclude the possibility that the increment of IL-6 contents in the conditioned medium for bupleuran 2IIc stimulated B cells is merely attributed to the increase in number of B cells, the IL-6 gene tran-

Fig. 3. Effect of bupleuran 2IIc on IL-6 production from normal B cells. Purified B cells from C3HrHeJ mice were cultured with or without bupleuran 2IIc Ž0.1 mgrml.. At the designated time points, IL-6 content in the culture supernatants was measured by ŽA. ELISA or ŽB. bioassay using MH-60 cell. Data were expressed as mean"S.D. Ž ns 4.. `: Control, v: bupleuran 2IIc.

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Fig. 4. Effect of bupleuran 2IIc on the transcription of IL-6 mRNA in normal B cells. The transcription of IL-6 mRNA was analyzed for 72-h cultured B cells with or without bupleuran 2IIc Ž0.1 mgrml. by RT-PCR method. Cyclophilin was used as a control.

natants at 72 h of early B cell lines were assayed for IL-6 production by ELISA. As shown in Fig. 5, bupleuran 2IIc had no effect on IL-6 production of SP2 or BCL 1-B20 cells, but bupleuran 2IIc enhanced the IL-6 secretion of Y16 cells. This result suggests that bupleuran 2IIc is a IL-6 inducer on normal murine B cells and certain B cell lines. 3.5. Structural requirement of bupleuran 2IIc for IL-6 production from normal B cells

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Endo-a-Ž1 4.-polygalacturonase digestion of bupleuran 2IIc gives a AramifiedB region ŽPG-1.,

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Fig. 6. Structural requirement of bupleuran 2IIc for IL-6 production from normal B cells. B cells Ž2=10 6 cellsrml. from C3HrHeJ mice were cultured for 3 days with or without bupleuran 2IIc Ž0.05 mgrml., PG-1, PG-2 or PG-3 Ž0.05 mgrml.. IL-6 content in the culture supernatants was measured by ELISA. Data were expressed as mean"SD Ž ns 4.. ) ) ) P - 0.001, ) P - 0.5 vs. control.

which is composed of rhamnogalacturonan core having side chains rich in neutral sugars, rhamnogalacturonan II-like region ŽPG-2. and oligogalacturonide fraction ŽPG-3. by gel filtration of Bio-Gel P30. When these carbohydrate fragments, which are obtained from the digest or bupleuran 2IIc, were assayed for IL-6 production in supernatants Žcultured for 3 days. of B cells by ELISA, only PG-1 enhanced the IL-6 production significantly and its effect was stronger than that of bupleuran 2IIc ŽFig. 6.. This result suggests that the AramifiedB region ŽPG-1. is the active site for IL-6 production by bupleuran 2IIc.

4. Discussion

Fig. 5. Effect of bupleuran 2IIc on IL-6 secretion from B cell line. B cell lines Y16, BCL 1 or SP2 Ž5=10 5 cellsrml. were separately cultured for 3 days in RPMI-1640 medium containing no FBS with or without bupleuran 2IIc Ž0.1 mgrml.. IL-6 content in the culture supernatants was measured by ELISA. Data were expressed as mean"S.D. Ž ns 4.. ) ) ) P - 0.001 vs. control.

Recently, we demonstrated that bupleuran 2IIc induced the proliferation and maturation of B cells and also enhanced the Ig secretion from B cells ŽSakurai et al., 1999.. However, its activation mechanism of B cells including its Ig secretion-enhancing activity has not been understood. Activated B cells secrete several cytokines such as IL-1, IL-6, IL-10 and nerve growth factor ŽNGF., which are involved

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in the regulation of growth and differentiation of B cells including Ig secretion ŽPistoia et al., 1986; Horii et al., 1988; O’Garra et al., 1988; Sung et al., 1988; Balkwill and Burke, 1989.. The present study showed that secreted IL-6 from B cells contributed to the enhancement of IgM production from bupleuran 2IIc-treated B cells as autocrine or paracrine systems. Because IgM production was reduced partially by anti-IL-6 antibody, other cytokines may also be involved in Ig secretion-enhancing activity of bupleuran 2IIc. In order to clarify the profiling of cytokine productions by bupleuran 2IIc-activated B cells, further experiments are now in progress by this research group. Bupleuran 2IIc activates B cells by T-cell-independent mechanism, indicating that bupleuran 2IIc is classified into T-cell-independent ŽTI. antigen. TI antigens are further divided into two categories, TI-1 and TI-2 ŽMond et al., 1983.. Generally, TI-2 antigens stimulate B cells by cross-linking of surface IgM ŽsIgM. as B cell receptor ŽBCR., and cytokines such as IL-1 andror IL-6 are required in several steps in the B cell responses, including differentiation into Ig-secreting cells and isotype switching ŽKishimoto and Hirano, 1988; O’Garra et al., 1988.. Because the addition of recombinant murine IL-6 enhanced the Ig secretion of bupleuran 2IIc-stimulated B cells ŽSakurai et al., 1999., bupleuran 2IIc seemed to be TI-2 antigen. However, the present study clearly showed that bupleuran 2IIc enhances the IL-6 production of B cells, and the resulting IL-6 enhances the Ig-secretion of B cell by an autocrine andror paracrine mechanism. Therefore, bupleuran 2IIc should be classified into category of TI-1 antigen rather than TI-2. LPS, which is known to be one of TI-1 antigens, activates B cells and differentiates B cells into Igsecreting cells ŽJacobs and Morrison, 1975; Tadakuma et al., 1982.. Bupleuran 2IIc enhances Ig secretion in splenic B cells of LPS-non-responder C3HrHeJ mouse as well as LPS-responder C3HrHeN mouse ŽSakurai et al., 1999.. C3HrHeJ mouse bears a defective lps gene, and recently, it was reported that the mechanism of defectiveness is due to a point mutation in the third exon of the Toll-like receptor-4 gene ŽTlr4 . ŽPoltorak et al., 1998; Hoshino et al., 1999; Qureshi et al., 1999.. These observations suggest that bupleuran 2IIc and LPS

may have different membrane receptors or different pathways of signal transduction on B cells. Although it has been shown that the AramifiedB region ŽPG-1. of bupleuran 2IIc acts as the active site of the polysaccharide to exert the mitogenic activity ŽSakurai et al., 1999., the present study clarified that the same region also acts as the active site of IL-6 secretion-enhancing activity on normal B cells. These results suggest that the mitogenic activity and IL-6 production-enhancing activity of bupleuran 2IIc might be exerted via same membrane receptor on B cell surface. It has been considered that polysaccharide antigens contain repeating structures due to the antigenic epitope, which are capable of cross-linking with membrane surface IgM ŽsIgM, BCR. in a multivalent fashion on the surface of a polysaccharide-responsible B cells, and activate B cells to induce proliferation by cross-linking ŽAndersson and Blomgren, 1971; Dorries et al., 1974.. Cross-linking of sIgM on B lymphocytes by anti-IgM also activates B cells and induces B cell proliferation, but does not induce Ig secretion in B cells ŽHobbs et al., 1991.. Bupleuran 2IIc stimulates B cells and not only induces proliferation but also enhances the secretion of IL-6 to differentiate into antibody-secreting cells. However, it is not known whether bupleuran 2IIc or AramifiedB region ŽPG-1. cross-links sIgM on B cells. If sIgMs on B cells are capable of cross-linking with bupleuran 2IIc, the Ig secretion-enhancing activity of bupleuran 2IIc may be exerted by not only merely cross-linking of sIgM on B cells, but also requiring costimulatory membrane receptor to transduce additional signals. Because bupleuran 2IIc has a complement-activating activity ŽYamada et al., 1989., complement receptors such as CD19rCD21 or CD21rCD35 also have a possibility to mediate additional signals. However, the receptor for bupleuran 2IIc and signal transductions on B cells are still unclear. In addition to normal murine spleen B cells, the enhanced IL-6 secretion by bupleuran 2IIc was also observed in B cell line, Y16. B cells were divided into two populations, B-1 and B-2. Y16 is characterized as early B cell, and is B-1 ŽCD5q. lineage ŽTominaga et al., 1989.. Because it has been reported that the B-1 subset is found predominantly in the peritoneal and pleural cavities but not in the

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spleen, it is not yet known whether bupleuran 2IIc enhances IL-6 secretion in all B cells in spleen or a part of population such as B-1 lineage cells. Further studies to clarify the population of B cells, which are activated by bupleuran 2IIc, are also being conducted by this research group. Recently, we demonstrated that bupleuran 2IIc was detected in the liver after oral administration to mice by using anti-polysaccharide antibody ŽSakurai et al., 1996., suggesting that a part of orally administered bupleuran 2IIc is incorporated into the circulation. IL-6 is a multifunctional cytokine and regulates various immune reactions ŽKishimoto 1989; Le and Vilcek, 1989.. Therefore, the IL-6 secretion-enhancing activity of B cells by bupleuran 2IIc may partially contribute in the immunomodulating activity of Chinese and Japanese herbal medicines containing B. falcatum L. To our knowledge, this is the first report that a plant polysaccharide enhances IL-6 secretion in B cells. Details on the interaction of bupleuran 2IIc with cell surface molecules on B cells and mode of action including signal transduction must await further study.

Acknowledgements We would like to thank Dr. M.H. Sakurai Žalso from our institute. for her useful discussions and Dr. M. Hayashi ŽCenter for Basic Research, The Kitasato Institute. for providing MH-60 cell, and Prof. Dr. K. Takatsu ŽInstitute of Medical Science, University of Tokyo. for providing the cell lines, Y16 and BCL1B20. This work was supported by a grant-in-aid of Research on Health Sciences Focusing on Drug Innovation from The Japan Health Sciences Foundation, from Japan Keirin Association, and Tsumura, Tokyo.

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