- Email: [email protected]
Induction of immunomodulating cytokines by a new polysaccharide–peptide complex from culture mycelia of Lentinus edodes
Immunopharmacology 40 Ž1998. 187–198
Induction of immunomodulating cytokines by a new polysaccharide–peptide complex from culture mycelia of Lentinus edodes Meiqin Liu b
a,)
, Jianzhong Li a , Fanzuo Kong a , Jiayou Lin b, Yang Gao
b
a Research Center for Eco-EnÕironmental Sciences, Chinese Academy of Sciences, Beijing 100085, China Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, China
Accepted 15 July 1998
Abstract The immunomodulating effect of a new polysaccharide–peptide complex from culture mycelia of Lentinus edodes ŽLE. was studied for elucidation of the mechanism of augmentation of cell-mediated immunity. RNA samples were isolated from the untreated and treated murine splenocytes and human peripheral blood mononuclear cells. RT-PCR was used to analyze the cytokine gene expression and bioassay was used to analyze the cytokine production. By administration of LE, the expression levels of IL-2 and TNF-a genes were augmented in the treated murine spleen mononuclear cells and human peripheral blood mononuclear cells. The production of IL-2 were augmented in the treated murine spleen mononuclear cells, and the production of TNF-a were augmented in the treated murine peritoneal exudate macrophages. The production of IL-2 and TNF-a were augmented in the treated human peripheral blood mononuclear cells. These results suggest that LE may induce Th immune responses. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Lentinus edodes; Polysaccharide–peptide complex; Induction of cytokines
Abbreviations: Ala, alanine; Arg, arginine; Asp, aspartic acid; Glu, glutamic acid; Gly, glycine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Tyr, tyrosine; Val, valine; cDNA, complementary deoxyribonucleic acid; ConA, conalbumin; CTL, cytotoxic T lymphocytes; DTH, delayed type hypersensitivity; EDTA, ethylenediaminetetraacetic acid; FCS, foetal calf serum; hIL-2, human IL-2; 1 H-NMR, proton-nuclear magnetic resonance; hTNF-a, human TNF-a; IL, interleukin; IR spectrum, infra-red spectrum; KS-2, polysaccharide from culture mycelia of Lentinus edode; LAK, lymphokineactivated killer; LE, polysaccharide–peptide complex from culture mycelia of Lentinus edodes; LNT, polysaccharide from Lentinus edodes fruit bodies; MMLV, moloney murine leukemia virus; mIL-2, murine IL-2; mTNF-a, murine TNF-a; NK, nature killer; PBS, phosphate buffered saline solution; PHA, phytohaemagglutinin; PBMC, peripheral blood mononuclear cells; PSK, polysaccharide complex from mycelia of Coriolus Õersicolor; PSPC, polysaccharide–protein complex from mycelia of Tricholoma lobayense; RNA, ribonucleic acid; RT-PCR, Reverse transcription polymerase chain reaction; TaqDNA polymerase, Thermus aquaticus DNA polymerase; TE buffer, Tris–EDTA buffer; TNF-a, tumor necrosis factor-a ) Corresponding author. Tel.: q86-10-62925511 ext. 2235; fax: q86-10-62555381; e-mail: [email protected] 0162-3109r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 8 . 0 0 0 4 3 - 5
188
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
1. Introduction The antitumor activity of various mushroom polysaccharides has been extensively investigated, such as: Lentinan ŽLNT., a high molecular weight having a backbone of b-Ž1 ™ 3.-D-glucan and side chains of both b-Ž1 ™ 3.-D- and b-Ž1 ™ 6.-D-linked D-glucose residues obtained from Lentinus edodes fruit bodies; Schizophyllan, a high molecular weight b-Ž1 ™ 3.Ž1 ™ 6.-D-glucan prepared from Schizophyllum commune culture filtrates; and PSK, a peptide containing b-Ž1 ™ 4.Ž1 ™ 3.-D- or b-Ž1 ™ 4.Ž1 ™ 6.-D-glucan extracted from Coriolus Õersidor culture mycelia, are known to exhibit antitumor activity ŽKomatsu et al., 1969; Sasaki and Takasuka, 1976; Tsukagoshi et al., 1984.. Administration of these compounds is known to inhibit the growth of various transplantable tumors in experimental animals and increase the survival rate. These compounds are considered to exert their antitumor activity through potentiation of the host animals’ defense system rather than direct inhibition of tumor cell growth ŽChihara et al., 1970; Maeda and Chihara, 1971.. LNT is well known as a type of biological response modifier ŽBRM. ŽHamuro and Chihara, 1985.. Augmentations of NK, CTL, LAK activities and DTH responses against tumor antigen were observed after administration of Lentinan ŽHamuro and Chihara, 1985; Suzuki et al., 1990.. These activities were thought to participate in the antitumor effects of LNT. Previous reports demonstrated that combined administration of LNT and IL-2 was able to induce endogenous LAK activity in both normal and tumor-bearing mice, and show the increased growth inhibitory effect and the significant prolongation of survival rate in MM46rC3HrHeN system. It offered a possible clinical application of a combination of LNT and IL-2 for immunotherapy against cancer without detrimental side effects ŽSuzuki et al., 1990.. KS-2, prepared by ethanol precipitation of the hot water extract of culture mycelia of L. edodes KSLE 007, suppressed the growth of Sarcoma 180 tumors and induced an interferon in mice when giving either orally or intraperitoneally ŽFujii et al., 1978.. Although LNT is thought to augment the immune response via modulation of the function of phagocytes such as macrophages, the precise mechanism for augmentation of cell-mediated immunity remains
to be elucidated ŽMaeda et al., 1971; Chihara et al., 1987.. As far as we know, there is no evidence that LNT and KS-2 induce gene expression in murine splenocytes, macrophages, and human PBMC for various cytokines in vitro and in vivo. We now report the effects of the administration of a new polysaccharide–peptide complex from culture mycelia of L. edodes ŽLE. on cytokine gene expression and production in vitro. After administration of LE, Ž1. the expression level and production of mIL-2 and mTNF-a were augmented compared with the control containing only the cells; Ž2. the expression level and production of hIL-2 and hTNF-a were augmented compared with the control containing only the cells. These results suggest that LE may induce Th immune responses.
2. Materials and methods 2.1. Characterization of polysaccharide–peptide complex from culture mycelia of L. edodes A loopful of L. edodes ACCC 50291 maintained on a cultivation medium agar slant was transferred to 100 ml of sterile medium in a 500-ml flask. After incubation on a reciprocating shaking for 10 days at 258C, the contents were centrifuged. The mycelia obtained were homogenized in a warring blender and subsequent extraction with boiling water was carried out three times at about 90–1008C. The hot water extracts were combined, dialyzed against water, then mixed with three volumes of ethanol and allowed to stand at 48C overnight. The precipitate was collected by centrifugation and washed with acetone. Then the powder of precipitates was dissolved in Tris–HCl, pH 7.0, dialyzed against the buffer and applied to a DEAE–Cellulose ŽDE 52 . column Ž3.5 cm = 30 cm., The column was eluted with 0.01 M Tris–HCl buffer. Six-milliliter aliquots of the effluent were collected by a fraction collector and analyzed for total sugar by the phenol-sulfuric acid method and for protein by Lowry method. The column was eluted until no sugar and protein were detected. Only one peak appeared and its corresponding fraction gave a white amorphous powder in a yield of 80% by ethanol
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
precipitation and centrifugation. Then it was rechromatographed on DEAE–Cellulose column, and gave a sharp single and symmetrical peak. This fraction was designated as LE. LE was proved to be homogenous by chromatography using DE 52 column, Sephadex G-100 gel filtration, UV spectroscopy and polyacrylamide gel electrophoresis etc. Identified by IR and UV spectroscopy, it was a polysaccharide– peptide complex, its molecular weight was about 5.08 = 10 5 Da by light diffusion method, and contained 94.2% carbohydrate and 5.8% protein. Complete acid hydrolysis revealed that its carbohydrate moiety was composed of sole glucose, and its peptide portion consisted of the following amino acids Žmol%., such as Asp Ž11.09%., Thr Ž6.16%., Ser Ž5.48%., Glu Ž12.66%., Pro Ž6.05%., Gly Ž5.55%., Ala Ž14.37%., Val Ž6.89%., Met Ž1.43%., Ile Ž4.70%., Leu Ž9.25%., Tyr Ž1.87%., Phe Ž3.81%., Lys Ž4.65%., His Ž1.24%. and Arg Ž4.77%.. The structure of carbohydrate moiety was determined by methylation, hydrolysis, acetylation, 1 H-NMR and IR spectrum, etc., indicating that it consisted of 2,4-di-O – Me – Glu, 2,4,6-tri-O – Me – Glu, and 2,3,4,6-tetra-O–Me–Glu, at a ratio of about 1:2:1 with b-D-glucose linkage ŽIR: 862 cmy1 and 928 cmy1 .. These suggested that LE had a b-Ž1 ™ 3.-Dglucan backbone with b-Ž1 ™ 6.-D-glucose side chains ŽLiu et al., 1998.. 2.2. Animals Male inbred BALBrc mice Ž8–12 weeks old. were purchased from the animal center, CAMS. The mice were housed under normal laboratory conditions, i.e., room temperature, 12r12 h light–dark cycle with free access to standard rodent chow and water. 2.3. Preparation of murine spleen mononuclear cells and peritoneal exudate macrophages The mice were sacrificed by cervical decapitation, the spleen cells were obtained by homogenizing spleens, and then the mononuclear cells were isolated by Ficoll–Hypaque gradient centrifugation. For bioassay of TNF-a, the peritoneal exudate macrophages were recovered by lavage of the peritoneal cavity with PBS.
189
For RNA extraction, the isolated mononuclear cells Ž3.6 = 10 6rml. were incubated in RPMI-1640 medium containing 10% FCS and different concentrations of LE Žcrude LE: 10, 20, 40 mgrml; LE: 2.5, 5, 10 mgrml.. The entry with 0 mg LErml was used as negative control, which contained only the cells, and PHA Ž10 mgrml. was used as positive control. The cells were cultured for 4 h at 378C, 5% CO 2 , in a humidified incubator. For bioassay of IL-2 and TNF-a, the cells Ž2 = 10 6rml. were cultured for 24 h. The entry with 0 mgrml was used as negative control, which contained only the cells, and PHA Ž25 mgrml. was used as positive control. The supernatants were harvested and stored at y708C.
2.4. Preparation of human peripheral blood mononuclear cells (PBMC) Human peripheral blood mononuclear cells were isolated from heparinised peripheral blood of different healthy donors by Ficoll–Hypaque gradient centrifugation, and cells from each donor were used in separate experiments. For RNA extraction, the isolated mononuclear cells Ž6 = 10 6rml. were incubated in RPMI-1640 medium containing 10% FCS and different concentrations of LE Ž0.5, 2.5, 12.5 mgrml.. The entry with 0 mgrml was used as negative control, which contained only the cells, and PHA Ž10 mgrml. was used as positive control. The cells were cultured for 4 h at 378C, 5% CO 2 , in a humidified incubator. For bioassay of IL-2 and TNF-a, the cells Ž2 = 10 6rml. were cultured for 24 h. The entry with 0 mgrml was used as negative control, which contained only the cells, and PHA Ž25 mgrml. was used as positive control. The supernatants were harvested and stored at y708C.
2.5. Bioassay of IL-2 and TNF-a The activity of IL-2 in supernatants was assayed using CTLL-2 cells bioassay ŽGillis et al., 1978; Tada et al., 1986.. The activity of TNF-a in the culture supernatants was assayed using L929 cells bioassay ŽRuff and Gifford, 1980; Green et al., 1984..
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
190
Table 1 List of polymerase chain reaction primers Genes
Primer sequences
hIL-2
5 -ACTCACCAGGATGCTCACAT X 3 -AGGTAATCCATCTGTTCAGA X 5 -TCTCGAACCCCGAGTGACAA X 3 -TATCTCTCAGCTCCACGCCA X 5 -GCATGGAGTCCTGTGGCAT X 3 -GGTGGCGTTTACGAAGATC X 5 -TGCAGCTCGCATCCTGTGTCA X 3 -AGAAGGCTATCCATCTCCTCA X 5 -GATGAGAAGTTCCCAAATGGCC X 3 -TGACGGCAGAGAGGAGGTTGAC X 5 -TGGGTCAGAAGGACTCCTATG X 3 -CAGGCAGCTCATAGCTCTTCT
hTNF-a hb-actin mIL-2 mTNF-a mb-actin
X
2.6. RNA extraction The mononuclear cells were lysed in 4 M guanidinium thiocyanate solution. Total RNA was extracted according to the single-step method previously described by Chomczynski and Sacchi Ž1987.. The RNA pellet was dissolved in 20 ml TE buffer, then electrophoresed on a 1% agarose gel and visual-
Expected size of PCR products Žbase pairs. 256 123 320 468 347 591
ized by ethidium bromide staining under ultraviolet light, it contained 28S and 18S. 2.7. ReÕerse transcription-polymerase chain reaction (RT-PCR) RNA sample was heated at 658C for 5 min and reverse-transcribed at 378C for 1 h in a 20 ml react-
Fig. 1. The linear conditions of RT-PCR. Ž1. Relationship between amounts of RNA and RT-PCR products of mTNF-a primers. M: PBR322rHinfl marker; the amounts of RNA: w1x 10 ng, w2x 100 ng, w3x 400 ng, w4x 600 ng, w5x 800 ng. Ž2. Relationship between numbers of cycles and RT-PCR products of mTNF-a primers. M: PBR322rHinfl marker; numbers of cycles: w1x 20, w2x 25, w3x 30, w4x 35, w5x 40. Representative results of three experiments are shown.
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
191
Fig. 2. RT-PCR analysis of cytokine gene expression in the murine spleen mononuclear cells Ž3.6 = 10 6 rml. inoculated with different concentrations of crude LE. The mean value Ž"S.E.M.. of three consecutive experiments is shown. ) p - 0.05 vs. control.
192 M. Liu et al.r Immunopharmacology 40 (1998) 187–198 Fig. 3. RT-PCR analysis of cytokine gene expression in the murine spleen mononuclear cells Ž3.6=10 6 rml. inoculated with different concentrations of LE. The mean value Ž"S.E.M.. of three consecutive experiments is shown. ) p- 0.05 vs. control.
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
ion mix containing 1.0 mg total RNA, 1.0 ml 12.5 mM mixture of all four deoxynucleotide triphosphates, 1 ml random hexmers Oligo ŽdT.12 – 18 ŽPromega, 100 ngrml., 4 ml 5 = RT buffer Ž250 mM Tris–HCl, pH 8.3, 375 mM KCl, 150 mM MgCl 2 ., and 1 ml MMLV reverse transcriptase Ž200 mnitsrml, Gibco-BRL, Bethesda, MD.. This solution was then heated at 958C for 5 min and quickly chilled on ice.
193
All PCR primer pairs specific for human and murine IL-2, TNF-a and b-actin were synthesized by DNA Synthesizer 381A ŽApplied Biosystems, Foster City, CA. in the Institute of Microbiology, Academia, Sinica, and the expected size of the PCR products are described in Table 1. The following components were added to the sample to make up 25 ml reaction mixture containing 2 ml cDNA, 2.5 ml TaqDNA polymerase buffer
Fig. 4. The effect of LE in different doses on mIL-2 and mTNF-a protein production. The cells Ž2 = 10 6 rml. were cultured for 24 h in the presence of LE, and the mIL-2 and mTNF-a protein was tested in cultured supernatants by bioassay. The mean value Ž"S.E.M.. of three consecutive experiments is shown. ) p - 0.05 vs. control.
194 M. Liu et al.r Immunopharmacology 40 (1998) 187–198 Fig. 5. RT-PCR analysis of cytokine gene expression in the human peripheral blood mononuclear cells Ž6=10 6 rml. inoculated with different concentrations of LE. The mean value Ž"S.E.M.. of three consecutive experiments is shown. ) p- 0.05 vs. control.
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
Ž100 mM Tris–HCl, pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 1 mgrml bovine serum albumin., 2.0 ml 2.5 mM mixture of all four deoxynucleotide triphosphates ŽPromega., 1 ml each of 5X and 3X primer Ž60 pmolrml., and 1 U TaqDNA polymerase Žthe Institute of Genetics, Academia Sinica.. PCR was performed for 35 cycles using a DNA thermal cycler Žthe Institute of Genetics, Academia Sinica.. Temperature cycling was initiated with each cycle as follows: Ž1. 958C for 40 s Ždenaturation.; Žb. 608C
195
for 1 min Žannealing.; Žc. 728C for 1 min Žextension.. Samples comprising 10 ml PCR products and molecular mass marker ŽPBR322rHinfl. were electrophoresed on a 1.2% agarose gel and visualized by ethidium bromide staining under ultraviolet light. 2.8. QuantitatiÕe RT-PCR Quantitative PCR was performed according to the method described by Rieckmann et al. Ž1994.. Den-
Fig. 6. The effect of LE in different doses on hIL-2 and hTNF-a protein production. The cells Ž2 = 10 6 rml. were cultured for 24 h in the presence of LE, and the hIL-2 and hTNF-a protein was tested in cultured supernatants by bioassay. The mean value Ž"S.E.M.. of three consecutive experiments is shown. ) p - 0.05 vs. control.
196
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
sitometric analysis was performed using a Pharmacia LKB Utroscan XL Laser densitometer. The values are given as ‘relative units’ and were calculated as follows: density of cytokine amplification productrdensity of b-actin amplification product= 100. 2.9. Statistics All experiments were repeated at least three times. Statistical analysis was performed by Student’s t-test. A confidence level of - 0.05 was considered significant ŽZar, 1984..
3. Results 3.1. Effects of LE on gene expression and production of cytokines in normal mice in Õitro We first examined the effects of administration of LE on cytokines gene expression in normal murine spleen mononuclear cells. Total RNA extracted from murine spleen mononuclear cells which were cultured with different concentrations of LE Žcrude LE: 10, 20, 40 mgrml; LE: 2.5, 5, 10 mgrml. was reverse-transcribed and amplified with cytokinespecific primers, as described in Section 2. Meanwhile, we used PHA ŽSigma, 10 mgrml., an immunestimulating agent, as positive control. In order to find out the proper condition of RTPCR, we studied the number of cycles and input RNA obtained from the positive control. As shown in Fig. 1, the assays are in the linear part of the curve. As shown in Figs. 2 and 3, the expression levels of IL-2, TNF-a genes were detected in the murine spleen mononuclear cells which were cultured with different concentrations of LE. We found that it augmented IL-2 gene expression compared with the control containing only the cells. We also found that LE augmented IL-2 protein production when the cells were cultured in the presence of 25, 50, 100 mgrml of LE ŽFig. 4.. This indicated that LE upregulated, in a dose dependent manner, IL-2 accumulation in the murine spleen mononuclear cells. In addition, we also found that LE enhanced the expres-
sion levels of TNF-a gene in the murine spleen mononuclear cells ŽFigs. 2 and 3. and TNF-a protein production in the peritoneal exudate macrophages which were cultured in the presence of 25, 50, 100 mgrml of LE ŽFig. 4.. These indicated that LE also up-regulated TNF-a accumulation in a dose dependent manner. These results raise the possibility that LE may induce Th immune responses. 3.2. Effects of LE on gene expression and production of cytokines in human peripheral blood mononuclear cells We further examined the effects of administration of LE on gene expression and production of cytokines in normal human peripheral blood mononuclear cells. Total RNA, extracted from human PBMC which were cultured with different concentrations of LE Ž0.5, 2.5, 12.5 mgrml., was reverse transcribed and amplified with cytokine-specific primers as described in Section 2. As shown in Fig. 5, the expression levels of IL-2 and TNF-a genes were detected in the human PBMC which were cultured with different concentrations of LE, indicating that LE enhanced the expression levels of IL-2 and TNF-a genes in a dose-dependent manner. We also found that when the cells were cultured in the presence of 25, 50, 100 mgrml of LE, it augmented the protein production of IL-2 and TNF-a ŽFig. 6.. This indicates that LE up-regulated IL-2 and TNF-a accumulation in the human PBMC in a dose dependent manner. These results further raise the possibility that LE may induce Th immune responses.
4. Discussion Our results are the first report describing the induction of cytokine gene expression and production in vitro by a new polysaccharide–peptide complex ŽLE. from culture mycelia of L. edodes ACCC 50291, which is different in origin and chemical properties compared with Lentinan and KS-2 ŽChihara et al., 1970; Fujii et al., 1978.. Lentinan is a high molecular weight polysaccharide having a backbone of b-Ž1 ™ 3.-D-glucan and side chains of
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
both b-Ž1 ™ 3.-D- and b-Ž1 ™ 6.-D-linked D-glucose residues obtained from L. edodes fruit bodies ŽSasaki and Takasuka, 1976.; KS-2 is mainly composed of a-linked mannose and a small amount of peptide consisting of serine, threonine and alanine with residual amounts of the other amino acids ŽFujii et al., 1978.. LE had a b-Ž1 ™ 3.-D-glucan backbone with b-Ž1 ™ 6.-D-glucose side chains, its peptide portion consisted of Asp, Glu, Ala and other amino acids. This suggests that LE is a new polysaccharide–peptide complex. By administration of LE, the expression levels of IL-2 and TNF-a genes were augmented in the treated murine spleen mononuclear cells and human peripheral blood mononuclear cells. The production of IL-2 were augmented in the treated murine spleen mononuclear cells, and the production of TNF-a were augmented in the treated murine peritoneal exudate macrophages. The production of IL-2 and TNF-a were augmented in the treated human peripheral blood mononuclear cells. Zhou et al. Ž1995. found that lentinan Ž1–125 mgrl. markedly enhanced ConA induced murine splenocytes proliferation and IL-2 production by murine splenocytes. Wang and Lin Ž1996. found that lentinan Ž=6. administration elicited release of TNF from macrophage in the presence of lipopolysaccharide ŽLPS.. In this study, the results suggest that LE may induce IL-2 and TNF-a in vitro both at the transcriptional level and protein level. It was suggested that there are a lot of immunomodulating cytokines and cytokine receptors in animals ŽAugus, 1992.. However, not all of immunomodulating cytokines were activated by antitumor drugs such as polysaccharides in vitro ŽHirose et al., 1990.. PSK consists of a heterogeneous mixture of glycosylated protein with an average molecular mass of approximately 100 KDa ŽTsukagoshi et al., 1984.. It has been reported that PSK induced gene expression in human peripheral blood mononuclear cells ŽPBMC. for IL-1a , IL-1b, IL-6, IL-8, TNF-a, etc. in vitro ŽHirose et al., 1990. and induced gene expression and production of TNFa and IL-8 in vivo by oral administration ŽKato et al., 1995.. PSPC and PSK only activated some immunomodulating cytokines, such as TNF-a, but most of immunomodulating cytokines were not affected in vivo ŽLiu et al., 1996.. In this study, we found that LE, a new polysaccharide–peptide complex, enhanced gene ex-
197
pression and production of IL-2 and TNF-a in vitro, suggesting that LE may induce Th immune responses. In order to investigate the overall antitumor effect of LE, the study on in vivo induction of gene expression and production of immunomodulating cytokines in the tumor-bearing mice is in process.
Acknowledgements The authors thank Miss Li Li for her technical assistance.
References Augus, W.T., 1992. The Cytokine Handbook. Academic Press, 368 pp. Chihara, G., Hamuro, J., Maeda, Y.Y., Arai, Y., Fukuoka, F., 1970. Fractionation and purification of the polysaccharides with marked antitumor activity, especially lentinan, from Lentinus edodes ŽBerk.. Sing Žan edible mushroom.. Cancer Res. 30, 2776–2781. Chihara, G., Suga, T., Hamuro, J., 1987. Antitumor and metastasis inhibitor, activities of lentinan as an immunomodulator. Cancer Detect. Pre. Suppl. 1, 423. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid quanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156. Fujii, T., Maeda, H., Suzuki, F., Ishida, N., 1978. Isolation and characterization of a new antitumor polysaccharide, KS-2, extracted from culture mycelia of Lentinus edodes. The Journal of Antibiotics XXXI Ž11., 1079–1090. Gillis, S., Ferm, M.M., Ou, W., Smith, K.A., 1978. T-cell growth factor: parameters of production and quantitative microassay for activity. J. Immunol. 120, 2027–2032. Green, L.M., Reade, J.L., Ware, C.F., 1984. Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines. Journal of Immunological Methods 70, 257–268. Hamuro, J., Chihara, G., 1985. Lentinan, an cell-oriented immunopotentiator: its experimental and clinical applications and possible mechanism of immune modulation. In: Fehiche, R.L. ŽEds.., Immune Modulation Agents and Their Mechanisms. Marcel Dekker, New York, pp. 409–435. Hirose, K., Claus, D.C.Z., Oppenheim, J.J., Matsushima, K., 1990. Induction of gene expression and production of immunomodulating cytokines by PSK in human peripheral blood mononuclear cells. Lymphokine Res. 4, 475. Kato, M., Hirose, K., Hakozaki, M., Ohno, M., Saito, Y., Izutani, R., Noguchi, J., Hori, Y., Okumota, S., Kuroda, D., Nomura,
198
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
H., Nishimatsu, S., Ohoyanagi, H., 1995. Induction of expression for immunomodulating cytokines in peripheral blood mononuclear cells in response to orally administered PSK, an immunomodulating protein-bound polysaccharide. Cancer Immunol. Immunother. 40, 152–156. Komatsu, N., Okubo, S., Kikumoto, S., Kimura, K., Saito, G., Sakai, S., 1969. Host mediated antitumor action of schizophyllan, a glucan produced by Schizophyllum commune. Gann. 60, 137–144. Liu, F., Fung, M.C., Ooi, V.E.C., Chang, S.T., 1996. Induction in the mouse of gene expression of immunomodulating cytokines by mushroom polysaccharide–protein complexes. Life Sciences 58 Ž21., 1795–1803. Liu, M.Q., Li, J.Z., Kong, F.Z., 1998. Purification, characterization, and Immunological function of a new polysaccharide– peptide complex from culture mycelia of Lentinus edodes. Acta Biochim. Biophys. Sinica Žin press.. Maeda, Y.Y., Chihara, G., 1971. Lentinan, a new immuno-accelerator of cell mediated responses. Nature 229, 634. Maeda, Y.Y., Hamuro, J., Chihara, G., 1971. The mechanisms of action of antitumor polysaccharides: I. The effects of antilymphocyte serum on the antitumor activity of lentinan. Int. J. Cancer 8, 41–46. Rieckmann, P., Albrecht, M., Kitze, B., Weber, T., Tumani, H., Broocks, A., Luer, W., Poser, S., 1994. Cytokine mRNA
levels in mononuclear blood cells from patients with multiple sclerosis. Neurology 44, 1523. Ruff, R.M., Gifford, E.G., 1980. Purification and physicochemical characterization of rabbit tumor necrosis factor. J. Immunol. 125, 1671–1677. Sasaki, T., Takasuka, N., 1976. Further study of the structure of lentinan, an antitumor polysaccharide from Lentinus edodes. Carbohydrate Research 47, 99–104. Suzuki, M., Higuchi, S., Taki, Y., Taki, S., Miwa, K., Hamure, J., 1990. Induction of endogenous lymphokine-activated killer activity by combined administration of lentinan and interleukin 2. Int. J. Immunopharmacol. 12 Ž6., 513–623. Tada, H., Shiho, O., Kuroshima, K., Koyama, M., Tsukamoto, K., 1986. An improved colorimetric assay for interleukin 2. Journal of Immunological Methods 93, 157–165. Tsukagoshi, S., Hashimoto, Y., Fujii, G., Kobayashi, H., Nomoto, K., Orita, K., 1984. Krestein ŽPSK.. Cancer Treat Rev. 11, 131. Wang, G.L., Lin, Z.B., 1996. The immunomodulatory effect of lentinan. Acta Pharmaceutica Sinica 31 Ž2., 86–90. Zar, J.H., 1984. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, NJ. Zhou, A.W., Ge, Z.D., Liang, J.S., Wang, B., Zheng, F.L., Li, C.Y., Cheng, M.Z., 1995. Immunomodulatory effects of lentinan. Chinese Pharmacological Bulletin 11 Ž2., 157–159.
Induction of immunomodulating cytokines by a new polysaccharide–peptide complex from culture mycelia of Lentinus edodes Meiqin Liu b
a,)
, Jianzhong Li a , Fanzuo Kong a , Jiayou Lin b, Yang Gao
b
a Research Center for Eco-EnÕironmental Sciences, Chinese Academy of Sciences, Beijing 100085, China Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100005, China
Accepted 15 July 1998
Abstract The immunomodulating effect of a new polysaccharide–peptide complex from culture mycelia of Lentinus edodes ŽLE. was studied for elucidation of the mechanism of augmentation of cell-mediated immunity. RNA samples were isolated from the untreated and treated murine splenocytes and human peripheral blood mononuclear cells. RT-PCR was used to analyze the cytokine gene expression and bioassay was used to analyze the cytokine production. By administration of LE, the expression levels of IL-2 and TNF-a genes were augmented in the treated murine spleen mononuclear cells and human peripheral blood mononuclear cells. The production of IL-2 were augmented in the treated murine spleen mononuclear cells, and the production of TNF-a were augmented in the treated murine peritoneal exudate macrophages. The production of IL-2 and TNF-a were augmented in the treated human peripheral blood mononuclear cells. These results suggest that LE may induce Th immune responses. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Lentinus edodes; Polysaccharide–peptide complex; Induction of cytokines
Abbreviations: Ala, alanine; Arg, arginine; Asp, aspartic acid; Glu, glutamic acid; Gly, glycine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Tyr, tyrosine; Val, valine; cDNA, complementary deoxyribonucleic acid; ConA, conalbumin; CTL, cytotoxic T lymphocytes; DTH, delayed type hypersensitivity; EDTA, ethylenediaminetetraacetic acid; FCS, foetal calf serum; hIL-2, human IL-2; 1 H-NMR, proton-nuclear magnetic resonance; hTNF-a, human TNF-a; IL, interleukin; IR spectrum, infra-red spectrum; KS-2, polysaccharide from culture mycelia of Lentinus edode; LAK, lymphokineactivated killer; LE, polysaccharide–peptide complex from culture mycelia of Lentinus edodes; LNT, polysaccharide from Lentinus edodes fruit bodies; MMLV, moloney murine leukemia virus; mIL-2, murine IL-2; mTNF-a, murine TNF-a; NK, nature killer; PBS, phosphate buffered saline solution; PHA, phytohaemagglutinin; PBMC, peripheral blood mononuclear cells; PSK, polysaccharide complex from mycelia of Coriolus Õersicolor; PSPC, polysaccharide–protein complex from mycelia of Tricholoma lobayense; RNA, ribonucleic acid; RT-PCR, Reverse transcription polymerase chain reaction; TaqDNA polymerase, Thermus aquaticus DNA polymerase; TE buffer, Tris–EDTA buffer; TNF-a, tumor necrosis factor-a ) Corresponding author. Tel.: q86-10-62925511 ext. 2235; fax: q86-10-62555381; e-mail: [email protected] 0162-3109r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 8 . 0 0 0 4 3 - 5
188
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
1. Introduction The antitumor activity of various mushroom polysaccharides has been extensively investigated, such as: Lentinan ŽLNT., a high molecular weight having a backbone of b-Ž1 ™ 3.-D-glucan and side chains of both b-Ž1 ™ 3.-D- and b-Ž1 ™ 6.-D-linked D-glucose residues obtained from Lentinus edodes fruit bodies; Schizophyllan, a high molecular weight b-Ž1 ™ 3.Ž1 ™ 6.-D-glucan prepared from Schizophyllum commune culture filtrates; and PSK, a peptide containing b-Ž1 ™ 4.Ž1 ™ 3.-D- or b-Ž1 ™ 4.Ž1 ™ 6.-D-glucan extracted from Coriolus Õersidor culture mycelia, are known to exhibit antitumor activity ŽKomatsu et al., 1969; Sasaki and Takasuka, 1976; Tsukagoshi et al., 1984.. Administration of these compounds is known to inhibit the growth of various transplantable tumors in experimental animals and increase the survival rate. These compounds are considered to exert their antitumor activity through potentiation of the host animals’ defense system rather than direct inhibition of tumor cell growth ŽChihara et al., 1970; Maeda and Chihara, 1971.. LNT is well known as a type of biological response modifier ŽBRM. ŽHamuro and Chihara, 1985.. Augmentations of NK, CTL, LAK activities and DTH responses against tumor antigen were observed after administration of Lentinan ŽHamuro and Chihara, 1985; Suzuki et al., 1990.. These activities were thought to participate in the antitumor effects of LNT. Previous reports demonstrated that combined administration of LNT and IL-2 was able to induce endogenous LAK activity in both normal and tumor-bearing mice, and show the increased growth inhibitory effect and the significant prolongation of survival rate in MM46rC3HrHeN system. It offered a possible clinical application of a combination of LNT and IL-2 for immunotherapy against cancer without detrimental side effects ŽSuzuki et al., 1990.. KS-2, prepared by ethanol precipitation of the hot water extract of culture mycelia of L. edodes KSLE 007, suppressed the growth of Sarcoma 180 tumors and induced an interferon in mice when giving either orally or intraperitoneally ŽFujii et al., 1978.. Although LNT is thought to augment the immune response via modulation of the function of phagocytes such as macrophages, the precise mechanism for augmentation of cell-mediated immunity remains
to be elucidated ŽMaeda et al., 1971; Chihara et al., 1987.. As far as we know, there is no evidence that LNT and KS-2 induce gene expression in murine splenocytes, macrophages, and human PBMC for various cytokines in vitro and in vivo. We now report the effects of the administration of a new polysaccharide–peptide complex from culture mycelia of L. edodes ŽLE. on cytokine gene expression and production in vitro. After administration of LE, Ž1. the expression level and production of mIL-2 and mTNF-a were augmented compared with the control containing only the cells; Ž2. the expression level and production of hIL-2 and hTNF-a were augmented compared with the control containing only the cells. These results suggest that LE may induce Th immune responses.
2. Materials and methods 2.1. Characterization of polysaccharide–peptide complex from culture mycelia of L. edodes A loopful of L. edodes ACCC 50291 maintained on a cultivation medium agar slant was transferred to 100 ml of sterile medium in a 500-ml flask. After incubation on a reciprocating shaking for 10 days at 258C, the contents were centrifuged. The mycelia obtained were homogenized in a warring blender and subsequent extraction with boiling water was carried out three times at about 90–1008C. The hot water extracts were combined, dialyzed against water, then mixed with three volumes of ethanol and allowed to stand at 48C overnight. The precipitate was collected by centrifugation and washed with acetone. Then the powder of precipitates was dissolved in Tris–HCl, pH 7.0, dialyzed against the buffer and applied to a DEAE–Cellulose ŽDE 52 . column Ž3.5 cm = 30 cm., The column was eluted with 0.01 M Tris–HCl buffer. Six-milliliter aliquots of the effluent were collected by a fraction collector and analyzed for total sugar by the phenol-sulfuric acid method and for protein by Lowry method. The column was eluted until no sugar and protein were detected. Only one peak appeared and its corresponding fraction gave a white amorphous powder in a yield of 80% by ethanol
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
precipitation and centrifugation. Then it was rechromatographed on DEAE–Cellulose column, and gave a sharp single and symmetrical peak. This fraction was designated as LE. LE was proved to be homogenous by chromatography using DE 52 column, Sephadex G-100 gel filtration, UV spectroscopy and polyacrylamide gel electrophoresis etc. Identified by IR and UV spectroscopy, it was a polysaccharide– peptide complex, its molecular weight was about 5.08 = 10 5 Da by light diffusion method, and contained 94.2% carbohydrate and 5.8% protein. Complete acid hydrolysis revealed that its carbohydrate moiety was composed of sole glucose, and its peptide portion consisted of the following amino acids Žmol%., such as Asp Ž11.09%., Thr Ž6.16%., Ser Ž5.48%., Glu Ž12.66%., Pro Ž6.05%., Gly Ž5.55%., Ala Ž14.37%., Val Ž6.89%., Met Ž1.43%., Ile Ž4.70%., Leu Ž9.25%., Tyr Ž1.87%., Phe Ž3.81%., Lys Ž4.65%., His Ž1.24%. and Arg Ž4.77%.. The structure of carbohydrate moiety was determined by methylation, hydrolysis, acetylation, 1 H-NMR and IR spectrum, etc., indicating that it consisted of 2,4-di-O – Me – Glu, 2,4,6-tri-O – Me – Glu, and 2,3,4,6-tetra-O–Me–Glu, at a ratio of about 1:2:1 with b-D-glucose linkage ŽIR: 862 cmy1 and 928 cmy1 .. These suggested that LE had a b-Ž1 ™ 3.-Dglucan backbone with b-Ž1 ™ 6.-D-glucose side chains ŽLiu et al., 1998.. 2.2. Animals Male inbred BALBrc mice Ž8–12 weeks old. were purchased from the animal center, CAMS. The mice were housed under normal laboratory conditions, i.e., room temperature, 12r12 h light–dark cycle with free access to standard rodent chow and water. 2.3. Preparation of murine spleen mononuclear cells and peritoneal exudate macrophages The mice were sacrificed by cervical decapitation, the spleen cells were obtained by homogenizing spleens, and then the mononuclear cells were isolated by Ficoll–Hypaque gradient centrifugation. For bioassay of TNF-a, the peritoneal exudate macrophages were recovered by lavage of the peritoneal cavity with PBS.
189
For RNA extraction, the isolated mononuclear cells Ž3.6 = 10 6rml. were incubated in RPMI-1640 medium containing 10% FCS and different concentrations of LE Žcrude LE: 10, 20, 40 mgrml; LE: 2.5, 5, 10 mgrml.. The entry with 0 mg LErml was used as negative control, which contained only the cells, and PHA Ž10 mgrml. was used as positive control. The cells were cultured for 4 h at 378C, 5% CO 2 , in a humidified incubator. For bioassay of IL-2 and TNF-a, the cells Ž2 = 10 6rml. were cultured for 24 h. The entry with 0 mgrml was used as negative control, which contained only the cells, and PHA Ž25 mgrml. was used as positive control. The supernatants were harvested and stored at y708C.
2.4. Preparation of human peripheral blood mononuclear cells (PBMC) Human peripheral blood mononuclear cells were isolated from heparinised peripheral blood of different healthy donors by Ficoll–Hypaque gradient centrifugation, and cells from each donor were used in separate experiments. For RNA extraction, the isolated mononuclear cells Ž6 = 10 6rml. were incubated in RPMI-1640 medium containing 10% FCS and different concentrations of LE Ž0.5, 2.5, 12.5 mgrml.. The entry with 0 mgrml was used as negative control, which contained only the cells, and PHA Ž10 mgrml. was used as positive control. The cells were cultured for 4 h at 378C, 5% CO 2 , in a humidified incubator. For bioassay of IL-2 and TNF-a, the cells Ž2 = 10 6rml. were cultured for 24 h. The entry with 0 mgrml was used as negative control, which contained only the cells, and PHA Ž25 mgrml. was used as positive control. The supernatants were harvested and stored at y708C.
2.5. Bioassay of IL-2 and TNF-a The activity of IL-2 in supernatants was assayed using CTLL-2 cells bioassay ŽGillis et al., 1978; Tada et al., 1986.. The activity of TNF-a in the culture supernatants was assayed using L929 cells bioassay ŽRuff and Gifford, 1980; Green et al., 1984..
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
190
Table 1 List of polymerase chain reaction primers Genes
Primer sequences
hIL-2
5 -ACTCACCAGGATGCTCACAT X 3 -AGGTAATCCATCTGTTCAGA X 5 -TCTCGAACCCCGAGTGACAA X 3 -TATCTCTCAGCTCCACGCCA X 5 -GCATGGAGTCCTGTGGCAT X 3 -GGTGGCGTTTACGAAGATC X 5 -TGCAGCTCGCATCCTGTGTCA X 3 -AGAAGGCTATCCATCTCCTCA X 5 -GATGAGAAGTTCCCAAATGGCC X 3 -TGACGGCAGAGAGGAGGTTGAC X 5 -TGGGTCAGAAGGACTCCTATG X 3 -CAGGCAGCTCATAGCTCTTCT
hTNF-a hb-actin mIL-2 mTNF-a mb-actin
X
2.6. RNA extraction The mononuclear cells were lysed in 4 M guanidinium thiocyanate solution. Total RNA was extracted according to the single-step method previously described by Chomczynski and Sacchi Ž1987.. The RNA pellet was dissolved in 20 ml TE buffer, then electrophoresed on a 1% agarose gel and visual-
Expected size of PCR products Žbase pairs. 256 123 320 468 347 591
ized by ethidium bromide staining under ultraviolet light, it contained 28S and 18S. 2.7. ReÕerse transcription-polymerase chain reaction (RT-PCR) RNA sample was heated at 658C for 5 min and reverse-transcribed at 378C for 1 h in a 20 ml react-
Fig. 1. The linear conditions of RT-PCR. Ž1. Relationship between amounts of RNA and RT-PCR products of mTNF-a primers. M: PBR322rHinfl marker; the amounts of RNA: w1x 10 ng, w2x 100 ng, w3x 400 ng, w4x 600 ng, w5x 800 ng. Ž2. Relationship between numbers of cycles and RT-PCR products of mTNF-a primers. M: PBR322rHinfl marker; numbers of cycles: w1x 20, w2x 25, w3x 30, w4x 35, w5x 40. Representative results of three experiments are shown.
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
191
Fig. 2. RT-PCR analysis of cytokine gene expression in the murine spleen mononuclear cells Ž3.6 = 10 6 rml. inoculated with different concentrations of crude LE. The mean value Ž"S.E.M.. of three consecutive experiments is shown. ) p - 0.05 vs. control.
192 M. Liu et al.r Immunopharmacology 40 (1998) 187–198 Fig. 3. RT-PCR analysis of cytokine gene expression in the murine spleen mononuclear cells Ž3.6=10 6 rml. inoculated with different concentrations of LE. The mean value Ž"S.E.M.. of three consecutive experiments is shown. ) p- 0.05 vs. control.
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
ion mix containing 1.0 mg total RNA, 1.0 ml 12.5 mM mixture of all four deoxynucleotide triphosphates, 1 ml random hexmers Oligo ŽdT.12 – 18 ŽPromega, 100 ngrml., 4 ml 5 = RT buffer Ž250 mM Tris–HCl, pH 8.3, 375 mM KCl, 150 mM MgCl 2 ., and 1 ml MMLV reverse transcriptase Ž200 mnitsrml, Gibco-BRL, Bethesda, MD.. This solution was then heated at 958C for 5 min and quickly chilled on ice.
193
All PCR primer pairs specific for human and murine IL-2, TNF-a and b-actin were synthesized by DNA Synthesizer 381A ŽApplied Biosystems, Foster City, CA. in the Institute of Microbiology, Academia, Sinica, and the expected size of the PCR products are described in Table 1. The following components were added to the sample to make up 25 ml reaction mixture containing 2 ml cDNA, 2.5 ml TaqDNA polymerase buffer
Fig. 4. The effect of LE in different doses on mIL-2 and mTNF-a protein production. The cells Ž2 = 10 6 rml. were cultured for 24 h in the presence of LE, and the mIL-2 and mTNF-a protein was tested in cultured supernatants by bioassay. The mean value Ž"S.E.M.. of three consecutive experiments is shown. ) p - 0.05 vs. control.
194 M. Liu et al.r Immunopharmacology 40 (1998) 187–198 Fig. 5. RT-PCR analysis of cytokine gene expression in the human peripheral blood mononuclear cells Ž6=10 6 rml. inoculated with different concentrations of LE. The mean value Ž"S.E.M.. of three consecutive experiments is shown. ) p- 0.05 vs. control.
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
Ž100 mM Tris–HCl, pH 8.3, 500 mM KCl, 15 mM MgCl 2 , 1 mgrml bovine serum albumin., 2.0 ml 2.5 mM mixture of all four deoxynucleotide triphosphates ŽPromega., 1 ml each of 5X and 3X primer Ž60 pmolrml., and 1 U TaqDNA polymerase Žthe Institute of Genetics, Academia Sinica.. PCR was performed for 35 cycles using a DNA thermal cycler Žthe Institute of Genetics, Academia Sinica.. Temperature cycling was initiated with each cycle as follows: Ž1. 958C for 40 s Ždenaturation.; Žb. 608C
195
for 1 min Žannealing.; Žc. 728C for 1 min Žextension.. Samples comprising 10 ml PCR products and molecular mass marker ŽPBR322rHinfl. were electrophoresed on a 1.2% agarose gel and visualized by ethidium bromide staining under ultraviolet light. 2.8. QuantitatiÕe RT-PCR Quantitative PCR was performed according to the method described by Rieckmann et al. Ž1994.. Den-
Fig. 6. The effect of LE in different doses on hIL-2 and hTNF-a protein production. The cells Ž2 = 10 6 rml. were cultured for 24 h in the presence of LE, and the hIL-2 and hTNF-a protein was tested in cultured supernatants by bioassay. The mean value Ž"S.E.M.. of three consecutive experiments is shown. ) p - 0.05 vs. control.
196
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
sitometric analysis was performed using a Pharmacia LKB Utroscan XL Laser densitometer. The values are given as ‘relative units’ and were calculated as follows: density of cytokine amplification productrdensity of b-actin amplification product= 100. 2.9. Statistics All experiments were repeated at least three times. Statistical analysis was performed by Student’s t-test. A confidence level of - 0.05 was considered significant ŽZar, 1984..
3. Results 3.1. Effects of LE on gene expression and production of cytokines in normal mice in Õitro We first examined the effects of administration of LE on cytokines gene expression in normal murine spleen mononuclear cells. Total RNA extracted from murine spleen mononuclear cells which were cultured with different concentrations of LE Žcrude LE: 10, 20, 40 mgrml; LE: 2.5, 5, 10 mgrml. was reverse-transcribed and amplified with cytokinespecific primers, as described in Section 2. Meanwhile, we used PHA ŽSigma, 10 mgrml., an immunestimulating agent, as positive control. In order to find out the proper condition of RTPCR, we studied the number of cycles and input RNA obtained from the positive control. As shown in Fig. 1, the assays are in the linear part of the curve. As shown in Figs. 2 and 3, the expression levels of IL-2, TNF-a genes were detected in the murine spleen mononuclear cells which were cultured with different concentrations of LE. We found that it augmented IL-2 gene expression compared with the control containing only the cells. We also found that LE augmented IL-2 protein production when the cells were cultured in the presence of 25, 50, 100 mgrml of LE ŽFig. 4.. This indicated that LE upregulated, in a dose dependent manner, IL-2 accumulation in the murine spleen mononuclear cells. In addition, we also found that LE enhanced the expres-
sion levels of TNF-a gene in the murine spleen mononuclear cells ŽFigs. 2 and 3. and TNF-a protein production in the peritoneal exudate macrophages which were cultured in the presence of 25, 50, 100 mgrml of LE ŽFig. 4.. These indicated that LE also up-regulated TNF-a accumulation in a dose dependent manner. These results raise the possibility that LE may induce Th immune responses. 3.2. Effects of LE on gene expression and production of cytokines in human peripheral blood mononuclear cells We further examined the effects of administration of LE on gene expression and production of cytokines in normal human peripheral blood mononuclear cells. Total RNA, extracted from human PBMC which were cultured with different concentrations of LE Ž0.5, 2.5, 12.5 mgrml., was reverse transcribed and amplified with cytokine-specific primers as described in Section 2. As shown in Fig. 5, the expression levels of IL-2 and TNF-a genes were detected in the human PBMC which were cultured with different concentrations of LE, indicating that LE enhanced the expression levels of IL-2 and TNF-a genes in a dose-dependent manner. We also found that when the cells were cultured in the presence of 25, 50, 100 mgrml of LE, it augmented the protein production of IL-2 and TNF-a ŽFig. 6.. This indicates that LE up-regulated IL-2 and TNF-a accumulation in the human PBMC in a dose dependent manner. These results further raise the possibility that LE may induce Th immune responses.
4. Discussion Our results are the first report describing the induction of cytokine gene expression and production in vitro by a new polysaccharide–peptide complex ŽLE. from culture mycelia of L. edodes ACCC 50291, which is different in origin and chemical properties compared with Lentinan and KS-2 ŽChihara et al., 1970; Fujii et al., 1978.. Lentinan is a high molecular weight polysaccharide having a backbone of b-Ž1 ™ 3.-D-glucan and side chains of
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
both b-Ž1 ™ 3.-D- and b-Ž1 ™ 6.-D-linked D-glucose residues obtained from L. edodes fruit bodies ŽSasaki and Takasuka, 1976.; KS-2 is mainly composed of a-linked mannose and a small amount of peptide consisting of serine, threonine and alanine with residual amounts of the other amino acids ŽFujii et al., 1978.. LE had a b-Ž1 ™ 3.-D-glucan backbone with b-Ž1 ™ 6.-D-glucose side chains, its peptide portion consisted of Asp, Glu, Ala and other amino acids. This suggests that LE is a new polysaccharide–peptide complex. By administration of LE, the expression levels of IL-2 and TNF-a genes were augmented in the treated murine spleen mononuclear cells and human peripheral blood mononuclear cells. The production of IL-2 were augmented in the treated murine spleen mononuclear cells, and the production of TNF-a were augmented in the treated murine peritoneal exudate macrophages. The production of IL-2 and TNF-a were augmented in the treated human peripheral blood mononuclear cells. Zhou et al. Ž1995. found that lentinan Ž1–125 mgrl. markedly enhanced ConA induced murine splenocytes proliferation and IL-2 production by murine splenocytes. Wang and Lin Ž1996. found that lentinan Ž=6. administration elicited release of TNF from macrophage in the presence of lipopolysaccharide ŽLPS.. In this study, the results suggest that LE may induce IL-2 and TNF-a in vitro both at the transcriptional level and protein level. It was suggested that there are a lot of immunomodulating cytokines and cytokine receptors in animals ŽAugus, 1992.. However, not all of immunomodulating cytokines were activated by antitumor drugs such as polysaccharides in vitro ŽHirose et al., 1990.. PSK consists of a heterogeneous mixture of glycosylated protein with an average molecular mass of approximately 100 KDa ŽTsukagoshi et al., 1984.. It has been reported that PSK induced gene expression in human peripheral blood mononuclear cells ŽPBMC. for IL-1a , IL-1b, IL-6, IL-8, TNF-a, etc. in vitro ŽHirose et al., 1990. and induced gene expression and production of TNFa and IL-8 in vivo by oral administration ŽKato et al., 1995.. PSPC and PSK only activated some immunomodulating cytokines, such as TNF-a, but most of immunomodulating cytokines were not affected in vivo ŽLiu et al., 1996.. In this study, we found that LE, a new polysaccharide–peptide complex, enhanced gene ex-
197
pression and production of IL-2 and TNF-a in vitro, suggesting that LE may induce Th immune responses. In order to investigate the overall antitumor effect of LE, the study on in vivo induction of gene expression and production of immunomodulating cytokines in the tumor-bearing mice is in process.
Acknowledgements The authors thank Miss Li Li for her technical assistance.
References Augus, W.T., 1992. The Cytokine Handbook. Academic Press, 368 pp. Chihara, G., Hamuro, J., Maeda, Y.Y., Arai, Y., Fukuoka, F., 1970. Fractionation and purification of the polysaccharides with marked antitumor activity, especially lentinan, from Lentinus edodes ŽBerk.. Sing Žan edible mushroom.. Cancer Res. 30, 2776–2781. Chihara, G., Suga, T., Hamuro, J., 1987. Antitumor and metastasis inhibitor, activities of lentinan as an immunomodulator. Cancer Detect. Pre. Suppl. 1, 423. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid quanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156. Fujii, T., Maeda, H., Suzuki, F., Ishida, N., 1978. Isolation and characterization of a new antitumor polysaccharide, KS-2, extracted from culture mycelia of Lentinus edodes. The Journal of Antibiotics XXXI Ž11., 1079–1090. Gillis, S., Ferm, M.M., Ou, W., Smith, K.A., 1978. T-cell growth factor: parameters of production and quantitative microassay for activity. J. Immunol. 120, 2027–2032. Green, L.M., Reade, J.L., Ware, C.F., 1984. Rapid colorimetric assay for cell viability: application to the quantitation of cytotoxic and growth inhibitory lymphokines. Journal of Immunological Methods 70, 257–268. Hamuro, J., Chihara, G., 1985. Lentinan, an cell-oriented immunopotentiator: its experimental and clinical applications and possible mechanism of immune modulation. In: Fehiche, R.L. ŽEds.., Immune Modulation Agents and Their Mechanisms. Marcel Dekker, New York, pp. 409–435. Hirose, K., Claus, D.C.Z., Oppenheim, J.J., Matsushima, K., 1990. Induction of gene expression and production of immunomodulating cytokines by PSK in human peripheral blood mononuclear cells. Lymphokine Res. 4, 475. Kato, M., Hirose, K., Hakozaki, M., Ohno, M., Saito, Y., Izutani, R., Noguchi, J., Hori, Y., Okumota, S., Kuroda, D., Nomura,
198
M. Liu et al.r Immunopharmacology 40 (1998) 187–198
H., Nishimatsu, S., Ohoyanagi, H., 1995. Induction of expression for immunomodulating cytokines in peripheral blood mononuclear cells in response to orally administered PSK, an immunomodulating protein-bound polysaccharide. Cancer Immunol. Immunother. 40, 152–156. Komatsu, N., Okubo, S., Kikumoto, S., Kimura, K., Saito, G., Sakai, S., 1969. Host mediated antitumor action of schizophyllan, a glucan produced by Schizophyllum commune. Gann. 60, 137–144. Liu, F., Fung, M.C., Ooi, V.E.C., Chang, S.T., 1996. Induction in the mouse of gene expression of immunomodulating cytokines by mushroom polysaccharide–protein complexes. Life Sciences 58 Ž21., 1795–1803. Liu, M.Q., Li, J.Z., Kong, F.Z., 1998. Purification, characterization, and Immunological function of a new polysaccharide– peptide complex from culture mycelia of Lentinus edodes. Acta Biochim. Biophys. Sinica Žin press.. Maeda, Y.Y., Chihara, G., 1971. Lentinan, a new immuno-accelerator of cell mediated responses. Nature 229, 634. Maeda, Y.Y., Hamuro, J., Chihara, G., 1971. The mechanisms of action of antitumor polysaccharides: I. The effects of antilymphocyte serum on the antitumor activity of lentinan. Int. J. Cancer 8, 41–46. Rieckmann, P., Albrecht, M., Kitze, B., Weber, T., Tumani, H., Broocks, A., Luer, W., Poser, S., 1994. Cytokine mRNA
levels in mononuclear blood cells from patients with multiple sclerosis. Neurology 44, 1523. Ruff, R.M., Gifford, E.G., 1980. Purification and physicochemical characterization of rabbit tumor necrosis factor. J. Immunol. 125, 1671–1677. Sasaki, T., Takasuka, N., 1976. Further study of the structure of lentinan, an antitumor polysaccharide from Lentinus edodes. Carbohydrate Research 47, 99–104. Suzuki, M., Higuchi, S., Taki, Y., Taki, S., Miwa, K., Hamure, J., 1990. Induction of endogenous lymphokine-activated killer activity by combined administration of lentinan and interleukin 2. Int. J. Immunopharmacol. 12 Ž6., 513–623. Tada, H., Shiho, O., Kuroshima, K., Koyama, M., Tsukamoto, K., 1986. An improved colorimetric assay for interleukin 2. Journal of Immunological Methods 93, 157–165. Tsukagoshi, S., Hashimoto, Y., Fujii, G., Kobayashi, H., Nomoto, K., Orita, K., 1984. Krestein ŽPSK.. Cancer Treat Rev. 11, 131. Wang, G.L., Lin, Z.B., 1996. The immunomodulatory effect of lentinan. Acta Pharmaceutica Sinica 31 Ž2., 86–90. Zar, J.H., 1984. Biostatistical Analysis. Prentice-Hall, Englewood Cliffs, NJ. Zhou, A.W., Ge, Z.D., Liang, J.S., Wang, B., Zheng, F.L., Li, C.Y., Cheng, M.Z., 1995. Immunomodulatory effects of lentinan. Chinese Pharmacological Bulletin 11 Ž2., 157–159.