0192-0561/92 $5.00 + .00 Pergamon Press Ltd. © 1992 International Society for Immunopharmacology.
Int. £ lmmunopharmac., Vol. 14, No. 5, pp. 821-830, 1992. Printed in Great Britain.
ENHANCEMENT OF MURINE ALVEOLAR MACROPHAGE FUNCTIONS BY ORALLY ADMINISTERED/3-GLUCAN TERUAKI SAKURAI,* KOICHI HASHIMOTO,* IWAO SUZUKI,* NAOHITO OHNO,* SHOZO OIKAWA,t AKIYOSHI MASUDAt and TOSHIRO YADOMAE*:~ *Laboratory of Immunopharmacology of Microbial Products, Tokyo College of Pharmacy, Horinouchi 1432-1, Hachioji, Tokyo 192-03; and tNippon Beet Sugar Mfg, Co., Ltd, Chuo-ku, Tokyo 104, Japan
(Received 22 August 1991 and in final form 27 December 1991)
- - The effect of orally administered SSG, a fl-l,3-glucan obtained from the culture filtrate of the fungus Sclerotinia sclerotiorum IFO 9395, on alveolar macrophage (AM) functions of CDF~ mice was examined. SSG administered orally (20, 40, 80 or 160 mg/kg) for I0 consecutive days enhanced the lysosomal enzyme activity of AM. The greatest enhancing effect was observed at 80 mg/kg of SSG. Multiple oral administrations of SSG (10 consecutive days) were needed to induce significant enhancing effects. Phagocytic activity and interleukin-1 (IL-1) production of AM were also augmented by oral administration of SSG, and the kinetics of the activated state differed depending on the kind of activity. However, H:O2 production of AM was not affected by SSG. Orally administered SSG also (40 or 80 mg/kg, 10 consecutive days) increased the number of AM and the greatest increment was observed 14 days after the first administration. On the other hand, the supernatant of Peyer's patch (PP) cells from mice administered SSG (80 mg/kg) orally stimulated the lysosomal enzyme activity of AM in vitro, and enhanced colony stimulating activity (CSA) was detected from this supernatant. These results demonstrate that SSG given by the oral route can activate murine AM both qualitatively and quantitatively, and it would mediated, at least in part, by the activation of PP cells in the intestine. Abstract
In recent years, biological response modifiers (BRMs) have been developed for cancer therapies and microbial infections. Much attention is being focused particularly on BRMs that can be administered orally (Hasegawa, Yoshikai, Okuda & Nomoto, 1990; Takeda, Yoshikai, Ohga & Nomoto, 1990), because some kinds of BRMs are known to be associated with significant toxicity when administered by parenteral routes (Peavy, Baughn & Musher, 1979; Berendt, Newborg & North, 1980; Maeda, Watanabe, Chihara & Rokutanda, 1984). Oral administration has the advantage of being easy to perform with essentially no accompanying pain or serious complications. Orally administered BRMs also enhanced the function of lymphoid organs in the intestine (Peyer's patches: PP) (Mfiller-Schoop & Good, 1975). It has been suggested that oral administration of BRMs induces good immune responses on mucosal sites.
fl-Glucans with 1,3 and/or 1,6 linkages are ubiquitous in nature as the major structural components of yeasts and fungi (Mullins, 1990) and are well known to possess immunomodulating effects and exhibit significant antitumor activities (Di Luzio, 1985). Clinically, two kinds of fl-l,3-glucans, lentinan (from Lentinus edodes) and schizophyllan (from Schizophyllum commune), have been applied as BRMs in Japan. We have also been studying the immunomodulating and antitumor activities of a highly branched fl-l,3-glucan, SSG, obtained from the liquid-cultured filtrate of the fungus Sclerotinia sclerotiorum IFO 9395 belonging to Ascomycotina (Suzuki, Hashimoto & Yadomae, 1988a; Hashimoto, Suzuki, Ohsawa, Oikawa & Yadomae, 1990). Interestingly, SSG exhibited antitumor and immunomodulating activities in mice when administered parenterally as well as orally (Suzuki, Hashimoto, Ohno, Tanaka & Yadomae,
*Author to whom correspondence should be addressed. 821
822
T. SAKURAIet al.
1989; Suzuki, Tanaka, Kinoshita, Oikawa, Osawa & Yadomae, 1990b; Hashimoto, Suzuki & Yadomae, 1991). Alveolar macrophages (AM) play an important role in controlling the pulmonary immune response because the lung is often exposed to various antigens in ambient air (Thepen, van Rooijen & Kraal, 1989). AMs are also important against infectious microorganisms and metastatic cancer cells in lung tissue (Nickerson & Jakab, 1990). Therefore, A M activation by BRMs is considered to be important in their antimicrobial and antimetastatic effects in the lung (Ogunbiyi, Conlon, Black & Eyre, 1988). AMs have several characteristics that differ from other local macrophages. These include iysosomal enzyme activity, metabolic activity for energy requirements, morphology, and surface markers because AMs are located at the a i r - t i s s u e interface (Horio, Ando, Ukeshima, Sugimoto & Tokumi, 1980; Akagawa, Maruyama, Takano, Kasai & Tokunaga, 1981; Akagawa & Tokunaga, 1985; Bilyk, Mackenzie, Papadimitriou & Holt, 1988). A few studies have reported that orally administered BRMs enhanced macrophage functions, particularly those of AMs (Perdig6n, de Macias, Alvarez, Oliver & de Ruiz Holgado, 1988; Florentin, Maral, de Sousa, Berardet, Hertz & Cloarec, 1989). Fidler et al. reported that oral administration of a lipophilic analog of muramyl dipeptide (MDP) activated both AMs and PMs, whereas parenteral (intravenous or intraperitoneal) administration activated only AMs or only PMs, respectively (Fidler, Fogler, Brownbill & Schumann, 1987). This suggested the possibility that BRMs administered orally can enhance the macrophage functions systemically. We also reported previously that orally administered SSG enhanced some functions of PM in mice (Suzuki et al., 1990b). Therefore, we concluded that investigations of the modulating effect on AM functions of orally administered SSG would be of value in enhancing our understanding of potential applications. In this study, we examined the effects of SSG administered orally on murine AM functions (lysosomal enzyme activity, phagocytic activity, H202 production, interleukin 1 (IL-1) production, increment of AM number) in relation to the activation of PP cells in the intestine. EXPERIMENTAL PROCEDURES
Animals Specific pathogen-free male CDF, (BALB/c × DBA/2), and C 3 H / H e J mice were purchased from
Japan SLC, Inc. (Shizuoka). CDF, mice were used at 6 - 8 weeks of age. C3H/HeJ mice were used at 4 weeks of age. These animals were bred under specific pathogen-free conditions. Preparation o f SSG The preparation method of SSG has been described previously (Suzuki et al., 1989). SSG contains less than 107o of protein and >98070 carbohydrate. Lipopolysaccharide (LPS) contamination of this preparation was less than 0.00014°70 (1.4 pg//ag of test sample). Alveolar macrophages Alveolar cells (AC) were harvested from CDF~ mice by bronchial lavage (Akagawa & Tokunaga, 1985). Mice were anesthesized by i.p. injection of 150 mg/kg of sodium pentobarbital (Nembutal ® , Dainabot Co., IL) and exsanguinated by cutting the arteria renalis. The thoracic cavity was opened and the lung removed with the trachea. The lavage fluid (Ca 2+ and Mg2+-free phosphate-buffered saline containing 0.05070 ethylenediamine tetraacetate) was introduced in 1.0 ml amounts from a syringe, the lung was gently massaged, and the fluid was withdrawn from the lungs into the syringe. A total of 6.0 ml of lavage fluid per mouse was used. AC were collected by centrifugation and resuspended in icecold RPMI-1640 medium (Nissui Seiyaku Co., Tokyo) supplemented with 5 mM N-[2-hydroxyethyl] piperazine-N' -[2-ethanesulfonic acid], penicillin G (100 U/ml), streptomycin (100/ag/ml) and 10070 heat-inactivated fetal calf serum (FCS) (Boehringer Mannheim). At least 97070 of AC were AM as judged by using cell smears of the Diff-Quik Stain kit (Kokusai Shiyaku Co., Hyogo) or the nonspecific esterase stain. AC were incubated for 2 h at 37°C in a CO2 incubator and were washed twice with warmed RPMI-1640 medium to remove any nonadherent cells. Peyer's patch cells The preparation method of PP cells has been described previously (Suzuki, Hashimoto & Yadomae, 1990a). Briefly, the PP were dissected out from CDF~ mice and placed in 8 ml of RPMI-1640 medium containing 5°70 heat-inactivated calf serum (Kyokuto Pharmaceutical Industries Co., Tokyo). The PP were placed on a slide, and were cut with a scalpel blade and teased gently with two glasses. The cell suspensions were passed through 8 0 - and 1 5 0 - g a g e sterile stainless steel sieves successively, and washed. The cells were resuspended in
Enhancement of Macrophage Functions appropriate volumes of RPMI-1640 medium containing 1007o heat-inactivated FCS. The viability of the cell suspensions thus obtained exceeded 95°7o. They always contained >9507o lymphocytes,
Int. £ lmmunopharmac., Vol. 14, No. 5, pp. 821-830, 1992. Printed in Great Britain.
ENHANCEMENT OF MURINE ALVEOLAR MACROPHAGE FUNCTIONS BY ORALLY ADMINISTERED/3-GLUCAN TERUAKI SAKURAI,* KOICHI HASHIMOTO,* IWAO SUZUKI,* NAOHITO OHNO,* SHOZO OIKAWA,t AKIYOSHI MASUDAt and TOSHIRO YADOMAE*:~ *Laboratory of Immunopharmacology of Microbial Products, Tokyo College of Pharmacy, Horinouchi 1432-1, Hachioji, Tokyo 192-03; and tNippon Beet Sugar Mfg, Co., Ltd, Chuo-ku, Tokyo 104, Japan
(Received 22 August 1991 and in final form 27 December 1991)
- - The effect of orally administered SSG, a fl-l,3-glucan obtained from the culture filtrate of the fungus Sclerotinia sclerotiorum IFO 9395, on alveolar macrophage (AM) functions of CDF~ mice was examined. SSG administered orally (20, 40, 80 or 160 mg/kg) for I0 consecutive days enhanced the lysosomal enzyme activity of AM. The greatest enhancing effect was observed at 80 mg/kg of SSG. Multiple oral administrations of SSG (10 consecutive days) were needed to induce significant enhancing effects. Phagocytic activity and interleukin-1 (IL-1) production of AM were also augmented by oral administration of SSG, and the kinetics of the activated state differed depending on the kind of activity. However, H:O2 production of AM was not affected by SSG. Orally administered SSG also (40 or 80 mg/kg, 10 consecutive days) increased the number of AM and the greatest increment was observed 14 days after the first administration. On the other hand, the supernatant of Peyer's patch (PP) cells from mice administered SSG (80 mg/kg) orally stimulated the lysosomal enzyme activity of AM in vitro, and enhanced colony stimulating activity (CSA) was detected from this supernatant. These results demonstrate that SSG given by the oral route can activate murine AM both qualitatively and quantitatively, and it would mediated, at least in part, by the activation of PP cells in the intestine. Abstract
In recent years, biological response modifiers (BRMs) have been developed for cancer therapies and microbial infections. Much attention is being focused particularly on BRMs that can be administered orally (Hasegawa, Yoshikai, Okuda & Nomoto, 1990; Takeda, Yoshikai, Ohga & Nomoto, 1990), because some kinds of BRMs are known to be associated with significant toxicity when administered by parenteral routes (Peavy, Baughn & Musher, 1979; Berendt, Newborg & North, 1980; Maeda, Watanabe, Chihara & Rokutanda, 1984). Oral administration has the advantage of being easy to perform with essentially no accompanying pain or serious complications. Orally administered BRMs also enhanced the function of lymphoid organs in the intestine (Peyer's patches: PP) (Mfiller-Schoop & Good, 1975). It has been suggested that oral administration of BRMs induces good immune responses on mucosal sites.
fl-Glucans with 1,3 and/or 1,6 linkages are ubiquitous in nature as the major structural components of yeasts and fungi (Mullins, 1990) and are well known to possess immunomodulating effects and exhibit significant antitumor activities (Di Luzio, 1985). Clinically, two kinds of fl-l,3-glucans, lentinan (from Lentinus edodes) and schizophyllan (from Schizophyllum commune), have been applied as BRMs in Japan. We have also been studying the immunomodulating and antitumor activities of a highly branched fl-l,3-glucan, SSG, obtained from the liquid-cultured filtrate of the fungus Sclerotinia sclerotiorum IFO 9395 belonging to Ascomycotina (Suzuki, Hashimoto & Yadomae, 1988a; Hashimoto, Suzuki, Ohsawa, Oikawa & Yadomae, 1990). Interestingly, SSG exhibited antitumor and immunomodulating activities in mice when administered parenterally as well as orally (Suzuki, Hashimoto, Ohno, Tanaka & Yadomae,
*Author to whom correspondence should be addressed. 821
822
T. SAKURAIet al.
1989; Suzuki, Tanaka, Kinoshita, Oikawa, Osawa & Yadomae, 1990b; Hashimoto, Suzuki & Yadomae, 1991). Alveolar macrophages (AM) play an important role in controlling the pulmonary immune response because the lung is often exposed to various antigens in ambient air (Thepen, van Rooijen & Kraal, 1989). AMs are also important against infectious microorganisms and metastatic cancer cells in lung tissue (Nickerson & Jakab, 1990). Therefore, A M activation by BRMs is considered to be important in their antimicrobial and antimetastatic effects in the lung (Ogunbiyi, Conlon, Black & Eyre, 1988). AMs have several characteristics that differ from other local macrophages. These include iysosomal enzyme activity, metabolic activity for energy requirements, morphology, and surface markers because AMs are located at the a i r - t i s s u e interface (Horio, Ando, Ukeshima, Sugimoto & Tokumi, 1980; Akagawa, Maruyama, Takano, Kasai & Tokunaga, 1981; Akagawa & Tokunaga, 1985; Bilyk, Mackenzie, Papadimitriou & Holt, 1988). A few studies have reported that orally administered BRMs enhanced macrophage functions, particularly those of AMs (Perdig6n, de Macias, Alvarez, Oliver & de Ruiz Holgado, 1988; Florentin, Maral, de Sousa, Berardet, Hertz & Cloarec, 1989). Fidler et al. reported that oral administration of a lipophilic analog of muramyl dipeptide (MDP) activated both AMs and PMs, whereas parenteral (intravenous or intraperitoneal) administration activated only AMs or only PMs, respectively (Fidler, Fogler, Brownbill & Schumann, 1987). This suggested the possibility that BRMs administered orally can enhance the macrophage functions systemically. We also reported previously that orally administered SSG enhanced some functions of PM in mice (Suzuki et al., 1990b). Therefore, we concluded that investigations of the modulating effect on AM functions of orally administered SSG would be of value in enhancing our understanding of potential applications. In this study, we examined the effects of SSG administered orally on murine AM functions (lysosomal enzyme activity, phagocytic activity, H202 production, interleukin 1 (IL-1) production, increment of AM number) in relation to the activation of PP cells in the intestine. EXPERIMENTAL PROCEDURES
Animals Specific pathogen-free male CDF, (BALB/c × DBA/2), and C 3 H / H e J mice were purchased from
Japan SLC, Inc. (Shizuoka). CDF, mice were used at 6 - 8 weeks of age. C3H/HeJ mice were used at 4 weeks of age. These animals were bred under specific pathogen-free conditions. Preparation o f SSG The preparation method of SSG has been described previously (Suzuki et al., 1989). SSG contains less than 107o of protein and >98070 carbohydrate. Lipopolysaccharide (LPS) contamination of this preparation was less than 0.00014°70 (1.4 pg//ag of test sample). Alveolar macrophages Alveolar cells (AC) were harvested from CDF~ mice by bronchial lavage (Akagawa & Tokunaga, 1985). Mice were anesthesized by i.p. injection of 150 mg/kg of sodium pentobarbital (Nembutal ® , Dainabot Co., IL) and exsanguinated by cutting the arteria renalis. The thoracic cavity was opened and the lung removed with the trachea. The lavage fluid (Ca 2+ and Mg2+-free phosphate-buffered saline containing 0.05070 ethylenediamine tetraacetate) was introduced in 1.0 ml amounts from a syringe, the lung was gently massaged, and the fluid was withdrawn from the lungs into the syringe. A total of 6.0 ml of lavage fluid per mouse was used. AC were collected by centrifugation and resuspended in icecold RPMI-1640 medium (Nissui Seiyaku Co., Tokyo) supplemented with 5 mM N-[2-hydroxyethyl] piperazine-N' -[2-ethanesulfonic acid], penicillin G (100 U/ml), streptomycin (100/ag/ml) and 10070 heat-inactivated fetal calf serum (FCS) (Boehringer Mannheim). At least 97070 of AC were AM as judged by using cell smears of the Diff-Quik Stain kit (Kokusai Shiyaku Co., Hyogo) or the nonspecific esterase stain. AC were incubated for 2 h at 37°C in a CO2 incubator and were washed twice with warmed RPMI-1640 medium to remove any nonadherent cells. Peyer's patch cells The preparation method of PP cells has been described previously (Suzuki, Hashimoto & Yadomae, 1990a). Briefly, the PP were dissected out from CDF~ mice and placed in 8 ml of RPMI-1640 medium containing 5°70 heat-inactivated calf serum (Kyokuto Pharmaceutical Industries Co., Tokyo). The PP were placed on a slide, and were cut with a scalpel blade and teased gently with two glasses. The cell suspensions were passed through 8 0 - and 1 5 0 - g a g e sterile stainless steel sieves successively, and washed. The cells were resuspended in
Enhancement of Macrophage Functions appropriate volumes of RPMI-1640 medium containing 1007o heat-inactivated FCS. The viability of the cell suspensions thus obtained exceeded 95°7o. They always contained >9507o lymphocytes,
