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GastroenterologiaJaponi~a Vol. 22, No. 5 Copyright 9 1987 by TheJapanese Society of Gastroenterology Printed in Japan --Original Article-- EFFECTS OF EXTRACT OF CULTURED I.ENTINUS EDODES MYCELIA (I.EM) ON POLYCLONAL ANTIBODY RESPONSE INDUCED BY POKEWEED MITOGEN Yasuhiro MIZOGUCHI, M.D.*, Hiroko KATOH, Ph.D.*, Kenzo KOBAYASHI, M.D.*, Sukeo YAMAMOTO, M.D.** and Seiji MORISAWA, M.D.*** *Third Department of Internal Medicine and ***First Department of Biochemistry, Osaka City University Medical School, Osaka 545,Japan, and **Osaka Socio-Medical Center Hospital, Osaka 557, Japan Summary When polyclonal antibody response induced by pokeweed mitogen (PWM) was estimated by measuring antibody-forming cells produced against trinitrophenylated sheep red blood cells (TNP-SRBC) using hemolytic plaque assay, it was found to be augmented by the extract of cultured Lentinus edodes mycelia (LEM). Since some factor enhancing antibody response was detected in the culture supernatant of LEM- treated macrophages, this was fractionated by gel filtration, which revealed a substance with a molecular weight of about 15,000 daltons. This suggested that interleukin.1 (IL-1) was produced, and that it had caused, at least partially, the enhancement of antibody response. This possibility was confirmed by the fftrect assay oflL-1 activity, which demonstrated increased DNA synthesis in PHA-stimulated thymocytes. Key Words: Antibody-formingcell, Extract of culturedLentinus edodesmycelia, Interleul~in-1. against viral infection. In particular, serocon- Introduction version from the HBe antigen-positive to the Various reports have suggested that HB HBe antibody-positive state is usually, thought virus-associated antigens are involved in cell- not always, associated with the termination of mediated immune responses in the mediation the HB virus infection and clearance of the o f liver cell damage, and that the expression HB virus or HBe antigen. Therefore, this sero- o f viral antigens on the cell m e m b r a n e ap- conversion may point to a favorable outcome pears to be a prerequisite for the immunologi- o f HBe antigen-positive chronic hepatitis and cal attack on hepatocytes. O n the other hand, may be used as a marker to evaluate the effects recent studies have emphasized that humoral o f clinical therapy. Some investigators have immune responses to viral antigens may play reported that the extract o f cultured Lentinus an important role in the defense mechanism edodes mycelia (LEM) is effective in the treatment of HBe antigen-positive chronic Received November 21, 1986. Accepted February 9, hepatitis, and that seroconversion occurs after 1987. treatment 1,2).A pharmaceutical preparation of Address for correspondence: Yasuhiro Mizoguchi, the water soluble fraction from the culture M.D., The Third Department of Internal Medicine, Osaka City University Medical School, 1-5-7Asahimachi, Abeno- medium of LEM has b e e n used, and it has ku, Osaka 545,Japan. b e e n reported to exhibit an immunomodula- 628 Y. MIZOGUCHI E T AL. Vol. 22, No. 5 tory effect in experimental studies3-5). In this culture medium (bagasse/defatted rice b r a n 5/1 w/w) report, we present our experimental results on mycelial pellet in suspension culture aseptically inocu- the effect of LEM on antibody response in lated vitro. 1 disrupted before the formation of fruit bodies Materials and Methods l incubated in water at 40-50~ for 6 hours 1. Preparation of LEM (autolysis and partial digestion of culture medium with The mushroom spores were germinated mycelial enzymes) 1 and cultured in a liquid medium containing 20 extracted with water at 60~ g of malt extract, 2.5 g of yeast extract and 2 g l of ammonium tartrate in 100 ml of medium. filtered and lyophilized 1 The resulting mycelial pellet was injected into pale brownish powder (LEM) a solid medium which was composed of (6 g'/kg medium) bagasse and defatted rice bran (5:1, w/w). The Fig, 1, Preparation of water soluble fraction from medium was disrupted before the formation culture medium of Lentinus edodes mycelia (LEM). of fruit bodies. The disrupted material was incubated in water at 40 to 50~ for 60 hours forming cells to promote autolysis of the mycelia and partial The mononuclear cell suspensions were digestion of the culture medium with mycelia incubated in vitro with 50/tg/ml of]0okeweed enzymes. The digest was then extracted with mitogen (PWM, Difco Co.) at 37~ for 72 water at 60~ The extract was aseptically hours in a humidified cell incubator with filtered and lyophilized, and the resulting aeration by 5% CO 2 in air. After incubation, pale-brownish powder was designated as TNP-SRBC were added to each mononuclear LEM. The yield of LEM was about 6 g/kg cell suspension, and the number of antibody- medium (Fig. 1). forming cells produced was counted by hemo- 2. Preparation of mononuclear cells from lytic plaque assay according to the method of human peripheral blood Jerne et al.Tk The effect of LEM on antibody Mononuclear cells were separated from the response was examined by adding various heparinized peripheral blood of healthy amounts of LEM simultaneously with PWM. adults by Ficoll-Conray density gradient cen- 5. Preparation of peritoneal exudate cells trifugation. The cells were washed twice and from normal guinea pigs suspended in Eagle's MEM containing 10% Twenty milliliters of sterilized Marcol 52 fetal calf serum to make a cell suspension of (Esso Oil Co.) was injected into the peritoneal 2 X 106 cells/ml. cavity of normal guinea pigs, and peritoneal 3. Preparation of trinitrophenylated sheep exudate cells were collected 4 days later by red blood cells (TNP-SRBC) perfusing the peritoneal cavity with 200 ml of Sheep red blood cells purchased from Japan Hank's solution. The oil phase was decanted, Biotest Co. were trinitrophenylated according and the aqueous phase was centrifuged at to the method of Rittenberg et al.6).After being 800• for 10 min in the cold. The cell pellets washed twice with Eagle's MEM, TNP-SRBC were washed three times with Hank's solution were suspended in the same medium at a and suspended in Eagle's MEM supplemented concentration of 7.5 X 108 cells/ml. with 10% fetal calf serum, 100 U/ml of peni- 4. Estimation of anti-TNP-SRBC antibody- cillin, and 100/~g/ml of streptomycin to make October 1987 Effects of LEM on polyclonal antibody response 629 incubated at 37~ for 48 hours. SH-thymidine (1 /tCi, specific activity 5 Ci/mmol) was also added, followed by another 24 hours of in- cubation. The radioactivity incorporated into the acid-soluble materials was then deter- mined by liquid scintillation spectrometry. Results 1. Effects of LEM on induction of antibody- forming cells Fig. 2. Sephadex G-75 column chromatography of When the peripheral blood mononuclear culture supernatantof LEM-treatedmacrophages. cells were stimulated in vitro with PWM in the presence of LEM, the number of anti-TNP- a cell suspension of 1 X 107 cells/ml. The cells SRBC plaque-forming cells (PFC) was signifi- were mostly macrophages except for some cantly greater than when they were stimulated contamination by lymphocytes and other cells. with PWM alone. When 200 ktg/ml of LEM 6. Fractionation of LEM-treated macrophage culture supernatant by gel filtration The culture supernatant of the LEM-treated macrophages was fractionated by gel filtration using a Sephadex G-75 column. The protein elution profile was obtained as shown in Fig. 2: Six fractions were collected separately, and each fraction was condensed to the original volume by uhrafiltration. The effect of these fractionated materials on antibody response was examined by adding them simultaneously with PWM to the PWM-induced polyclonal antibody production system described above. 7. Assay of interleukin-1 ( I L l ) activity in LEM-treated macrophage culture superna- tant IL-1 activity was measured by thymocyte proliferation assay according to the method of Simon et al3 I. Thymocytes prepared from C 3 H / H e J mice (male, 6 weeks old) were suspended in RPMI 1640 solution supple- mented with 5% fetal calf serum and 5 X 10-5 M 2-mercaptoethanol to make a cell suspen- sion of 2 X 10e cells/ml. Various volumes of the culture supernatant of the LEM-treated macrophages and 2/2g/ml of PHA were added Fig. 3. Effectsof LEM on PWM-inducedPFC. to the thymocyte suspensions, and these were n=10, *p<0.01 630 Y. MIZOGUCHI E T AL. Vol. 22, No. 5 was added simultaneously with PWM, PFC c o m p a r e d with w h e n the L E M - u n t r e a t e d production increased m o r e than twofold (Fig. m a c r o p h a g e culture supernatant was added 3). (Fig. 4). 2. Effects of LEM-treated macrophage cul- 3. Fractionation o f LEM-treated m a c r o p h a g e ture supernatant on induction of anti- culture supernatant body-forming cells In an attempt to isolate the active materials T o study the possible m e c h a n i s m by which in the culture supernatant of the LEM-treated LEM enhances antibody response, we ana- macrophages, the culture fluid was frac- lyzed its effect on m a c r o p h a g e activity, since tionated into six fractions, and the n u m b e r o f activated macrophages affect antibody produc- anti-SRBC PFC was determined. As a result, it tion through the production of IL-1 a n d / o r was found that the fifth fraction was the most increased expression o f Ia antigen on the cell potent in e n h a n c i n g antibody response (Fig. surface. W h e n the culture supernatant pre- 5). p a r e d from the LEM-treated macrophages was T h e elution profile indicated that the frac- a d d e d to the m o n o n u c l e a r cells with PWM, tion contained an active substance having a the n u m b e r of PFC was significantly elevated molecular weight o f about 15,000 daltons. This suggested that IL-1 was produced from the macrophages by activation with LEM. 4. IL-1 activity in LEM-stimulated macro- Fig. 4. Effectsof LEM-sfimulatedmacrophage culture Fig. 5. Effects of subfracfions of LEM-treated macro- supernatant on PWM-induced PFC. phage culture supematant on PFC. n=10, *p<0.01 n=10, *p<0.01 October 1987 Effects of LEM on polyclonal antibody response 631 added to the LEM-treated macrophage culture supernatant, IL-1 activity was not detected at all. Discussion LEM has been known to exhibit an anti- tumor effect in experimental studies 9-a6), and some reports have shown that seroconversion occurs after treatment in patients with HBe antigen-positive chronic hepatitis 1). Further- more, a multicentered, o p e n study trial has b e e n performed to evaluate the effectiveness and safety o f LEM on HBe antigen-positive chronic hepatitis 2). As a result, it has been concluded that LEM is useful for ameliorating the HBeAg/anti-HBe system, and improve- ments in liver function test parameters espe- cially transaminase levels have been observed. T h e functional mechanism by which LEM exhibits its therapeutic effect mostly remains to be elucidated, although some experimental and clinical studies have suggested that LEM might possess immunomodulatory activity~). Fig. 6. Effectsof LEM-stimulatedmacrophage culture T h e experimental results of the present supernatant on SH-thymidineuptake of PHA-sfimu- lated thymocytes. study indicated that LEM e n h a n c e d antibody n=10, *p<0.01 response, and that this may have been at- tributable, at least partially, to its capacity for phage culture supernatant macrophage activation. This was because IL-1 activity in the culture supernatant o f antibody response was augmented by adding the LEM-treated macrophages was confirmed LEM, and IL-1 production increased when the by examining its effect o n DNA synthesis cells were stimulated with LEM. IL-l-mediated i n d u c e d by PHA stimulation. When the induction of antibody production has been thymocytes were stimulated with PHA in the reported before aT). It was also found that the presence o f the culture supernatant o f the effect o f LEM on antibody response was LEM-treated macrophages, DNA synthesis was d e p e n d e n t on the dose. Thus, the most effec- significantly increased compared with when tive concentration was 200/~g/ml, and larger the L E M - u n t r e a t e d m a c r o p h a g e culture amounts were not necessarily beneficial. The supernatant was added (Fig. 6). In addition, reason for this is not clear at the present stage, IL-1 activity was confirmed using IL-1 mono- but it is assumed that it may be the optimal clonal antibody. When more than 10/A (10/xg concentration to induce the suitable modula- protein/ml) o f IL-1 monoclonal antibody tion o f the macrophages. Since our experi- (donated by the Laboratory o f Cellular Tech- ments reported here were carried out in vitro, nology, Ohtsuka Pharmaceutical Co. Ltd.) was further investigations including in vivo ex- 632 E MIZOGUCHI E T AL. Vol. 22, No. 5 p e r i m e n t s a r e u n d o u b t e d l y n e c e s s a r y to i n t e r - pulmonary immune function: Physicochemical char- pret and evaluate the clinical effect of LEM on acterization of two distinct species of lymphocyte- activating factor produced by rabbit alveolar macro- immune responses. phages.J Immunol 1981; 126:1534 9) Chihara G, et al: Inhibition of mouse sarcoma 180 by References polysaccharides from Lentinus edodes (Berk.) Sing. Nature 1969; 222:687 1) Amagase H, et al: Treatment of hepatitis B patient 10) Chihara G, et al: Fractionation and purification of the with Lentinus edodes mycelia (LEM). 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