Eur J Clin Pharmacol (l 992) 42:623-628 European J . . . . .
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© Springer-Verlag 1992
In vitro generation of activated natural killer cells and cytotoxic macrophages with lentinan M. Tani, H. Tanimura, H. Yamaue, M. Iwahashi, T. Tsunoda, M. Tamai, K. Noguchi, and K. Arii Department of Gastroenterological Surgery, Wakayama Medical College, Wakayama, Japan Received: August 20,1991/Accepted in revised form: January 4, 1992
Summary. The in vitro effect of lentinan in inducing activation of killer cells and cytotoxic macrophages has been examined. H u m a n peripheral blood mononuclear cells were cultured with lentinan for 2, 4 and 8 days. After 4days cytotoxicity was increased 4% by lentinan < 1,000 ng/ml. After 8 days, it was increased 12% by 25 and 1,000 ng/ml lentinan. The phenotype of the killer cells induced by lentinan was C D 2 + , C D 1 6 + and CD56 +, suggesting that they were natural killer cells. Macrophages separated from the spleens of 6 patients with gastric cancer were cultured with lentinan for 7 days, and their cytotoxicity increased 19%. The optimal concentration of lentinan was from 25 to 100 ng/ml. The findings suggest that the antitumour effect of lentinan is due to the activation of killer cells in vivo, because the optimal concentration of lentinan for the induction of killer cells in vitro was equivalent to the plasma concentration obtained after clinical doses of this agent.
Key words: Lentinan, Natural killer cell; cytolytic activity, cytotoxic macrophage, in vitro The role of interleukin-2 (IL-2) in the augmentation of natural killer (NK) cell activity has been demonstrated [1, 2]. The augmentation of NK cell activity involves a collaboration between IL-2 and interferon-}'(IFN-~), in which IL-2 stimulates mononuclear cells to produce IFN-7[3]. IFN, in addition to its antiviral activity, has been found to have a role in the regulation of N K cell [1, 4, 5] and macrophage [6] activity. Lentinan, which has been used as a potent biological response modifier (BRM) in cancer therapy, is derived from Lentinus edodus. It fails to directly suppress tumour cell growth in vitro [7-9], but augmentation of antitumour immunity by B R M is important to obtain a clinical response, because antitumour immunity in cancer patients is suppressed, as shown by delayed type hypersensitivity skin testing [10] and the autologous mixed lymphocyte reaction ( A M L R ) [11]. Investigating in vitro effects of B R M is neccesary to comprehend why the B R M brings clinical benefit. However, the in vitro effects of lentinan have
never been reported whereas various accounts related with its in vivo effects have been published [7]. The activity of N K cells and cytotoxic macrophages has now been evaluated following stimulation with lentinan in vitro.
Experimental procedures
Drugs Lentinan is a polysaccharide prepared from Lentinus edodes, which has an in vivo antitumour effect [8].It was provided by the Yamanouchi Pharmaceutical Co. (Tokyo, Japan).
Induction of effector cells by lentinan Peripheral blood mononuclear cells (PBMC) were obtained from 6 healthy subjects and 14 cancer patients (9 with gastric cancer, 2 with colon cancer, 2 with pancreatic cancer, and 1 with rectal leiomyosarcoma) by Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) gradient centrifugation (400 g for 30 rain, 20°C). PBMC were suspended in RPMI-1640 medium (Nissui Co., Tokyo, Japan) supplemented with 2 mM L-glutamine, 100 U. ml-1 penicillin, 100 pg-ml-1 streptomycin, 50 gM 2-mercaptoethanol, and 10% heat-inactivated human AB serum (complete medium). Mononuclear cells were separated from the spleens of 6 patients with gastric cancer and were cultured on 10% auto-serum coated Petri dishes (Falcon, No. 3003), for 1 h, at 37 °C, in a humidified 5% CO~ atmosphere. After the incubation, the nonadherent cells were removed by washing and the adherent cells were incubated for a further 16 h to exclude dendritic cells. Finally, the adherent cells were harvested, suspended in complete medium, and used as splenic macrophages in the subsequent studies. Lentinan was suspended in complete medium at concentrations of 1-2,000 ng. ml-1, and PBMC and splenic macrophages were suspended in the same medium at 2 x 106 cells, ml z.For in vitro stimulation, PBMC were incubated with lentinan (1-2,000 ng. ml- 1) for 2, 4, or 8 days, at 37°C, in a humidified 5% CO2 atmosphere, and splenic macrophages were incubated with lentinan 1-1,000ng.ml i for 8 days under the same conditions.
Target cells The target cells were the K562, chronic myelogenous leukemia cell line. Cells were maintained in medium with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA).
624 %
Determination of the serum concentration of lentinan in cancer patients
J
60
After the intravenous infusion of lentinan (1 mg), samples were collected and the serum lentinan concentration was determined using the Toxicolor test (Seikagaku Kogyo, Tokyo, Japan) [12].
Results /.0
Optimal dose of lentinan and optimal incubation period for the induction of killer cells
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Lentinan (-)
Lentinan (,)
Fig.1. Cytotoxicity of PBMC against K562 cells after stimulation with lentinan in vitro
After 2 days of culture of PBMC with lentinan 12,000 ng.m1-1, cytotoxicity was not increased (Fig. 1). However, after 4 days of culture, an increase in cytotoxicity was observed (P < 0.01), and the concentration of lentinan that augmented cytotoxic activity was always less than 1,000 ng. ml - ~(Fig. 2). After 8 days of culture, the cytotoxicity was 11.9% of the maximal increase (P < 0.01; Fig. 3), and the optimal lentinan concentration was below 1,000 ng. ml- 1. The dose titration of lentinan for cytotoxic activity is shown in Fig. 4. The optimal concentration of lentinan showed 2 peaks, one from 5 to 25 ng. ml-1 and 1,000 ng. ml- 1for the 8-day culture (P < 0.01, Fig. 4).
Induction of cytotoxicmacrophages by lentinan Cytotoxic assay A 4-h 5~Cr-release assay was performed for killer cells and a 16-h ~25Irelease assay was performed for cytotoxic macrophages. The target cells were labelled for 1 h at 37 °C with 100 gCi Na251CrO4 or with 125Ideoxyuridine for I h at 37 °C, and then washed three-times with medium containing 10% FCS. Then, 100 ~tl 51Cr-labeled tumour cells (1 x 10S/ml) was added in triplicate to 100 ~tl effector cells (the effector-to-target ratio was fixed at 15:1) in round-bottomed microtitre plates (Corning No.25850). After a 4-h incubation for killer cells, and a 16-h incubation for macrophages, both at 37 °C, the supernatants were harvested and the radioactivity was counted with a gamma counter. Spontaneous release was determined by the incubation of labelled target cells in complete medium alone, and did not exceed 10% of the maximum release following the addition of 1 M HC1. The percentage specific cytotoxicity was calculated as; (all 51Cr or ~25Ivalues were in cpm): test release-spontaneous release x 100 maximum release-spontaneous release
The cytotoxicity of splenic macrophages for K562 cells was increased from 0.5% to 51.5% (mean 18.8% ) by lentinan (P < 0.05; Fig. 5), and the optimal concentration of lentinan was 25 to 250 ng. ml- 1, similar to that for PBMC (Fig. 6). % P
Iof ( ~ [ ~ ( ~
© Springer-Verlag 1992
In vitro generation of activated natural killer cells and cytotoxic macrophages with lentinan M. Tani, H. Tanimura, H. Yamaue, M. Iwahashi, T. Tsunoda, M. Tamai, K. Noguchi, and K. Arii Department of Gastroenterological Surgery, Wakayama Medical College, Wakayama, Japan Received: August 20,1991/Accepted in revised form: January 4, 1992
Summary. The in vitro effect of lentinan in inducing activation of killer cells and cytotoxic macrophages has been examined. H u m a n peripheral blood mononuclear cells were cultured with lentinan for 2, 4 and 8 days. After 4days cytotoxicity was increased 4% by lentinan < 1,000 ng/ml. After 8 days, it was increased 12% by 25 and 1,000 ng/ml lentinan. The phenotype of the killer cells induced by lentinan was C D 2 + , C D 1 6 + and CD56 +, suggesting that they were natural killer cells. Macrophages separated from the spleens of 6 patients with gastric cancer were cultured with lentinan for 7 days, and their cytotoxicity increased 19%. The optimal concentration of lentinan was from 25 to 100 ng/ml. The findings suggest that the antitumour effect of lentinan is due to the activation of killer cells in vivo, because the optimal concentration of lentinan for the induction of killer cells in vitro was equivalent to the plasma concentration obtained after clinical doses of this agent.
Key words: Lentinan, Natural killer cell; cytolytic activity, cytotoxic macrophage, in vitro The role of interleukin-2 (IL-2) in the augmentation of natural killer (NK) cell activity has been demonstrated [1, 2]. The augmentation of NK cell activity involves a collaboration between IL-2 and interferon-}'(IFN-~), in which IL-2 stimulates mononuclear cells to produce IFN-7[3]. IFN, in addition to its antiviral activity, has been found to have a role in the regulation of N K cell [1, 4, 5] and macrophage [6] activity. Lentinan, which has been used as a potent biological response modifier (BRM) in cancer therapy, is derived from Lentinus edodus. It fails to directly suppress tumour cell growth in vitro [7-9], but augmentation of antitumour immunity by B R M is important to obtain a clinical response, because antitumour immunity in cancer patients is suppressed, as shown by delayed type hypersensitivity skin testing [10] and the autologous mixed lymphocyte reaction ( A M L R ) [11]. Investigating in vitro effects of B R M is neccesary to comprehend why the B R M brings clinical benefit. However, the in vitro effects of lentinan have
never been reported whereas various accounts related with its in vivo effects have been published [7]. The activity of N K cells and cytotoxic macrophages has now been evaluated following stimulation with lentinan in vitro.
Experimental procedures
Drugs Lentinan is a polysaccharide prepared from Lentinus edodes, which has an in vivo antitumour effect [8].It was provided by the Yamanouchi Pharmaceutical Co. (Tokyo, Japan).
Induction of effector cells by lentinan Peripheral blood mononuclear cells (PBMC) were obtained from 6 healthy subjects and 14 cancer patients (9 with gastric cancer, 2 with colon cancer, 2 with pancreatic cancer, and 1 with rectal leiomyosarcoma) by Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) gradient centrifugation (400 g for 30 rain, 20°C). PBMC were suspended in RPMI-1640 medium (Nissui Co., Tokyo, Japan) supplemented with 2 mM L-glutamine, 100 U. ml-1 penicillin, 100 pg-ml-1 streptomycin, 50 gM 2-mercaptoethanol, and 10% heat-inactivated human AB serum (complete medium). Mononuclear cells were separated from the spleens of 6 patients with gastric cancer and were cultured on 10% auto-serum coated Petri dishes (Falcon, No. 3003), for 1 h, at 37 °C, in a humidified 5% CO~ atmosphere. After the incubation, the nonadherent cells were removed by washing and the adherent cells were incubated for a further 16 h to exclude dendritic cells. Finally, the adherent cells were harvested, suspended in complete medium, and used as splenic macrophages in the subsequent studies. Lentinan was suspended in complete medium at concentrations of 1-2,000 ng. ml-1, and PBMC and splenic macrophages were suspended in the same medium at 2 x 106 cells, ml z.For in vitro stimulation, PBMC were incubated with lentinan (1-2,000 ng. ml- 1) for 2, 4, or 8 days, at 37°C, in a humidified 5% CO2 atmosphere, and splenic macrophages were incubated with lentinan 1-1,000ng.ml i for 8 days under the same conditions.
Target cells The target cells were the K562, chronic myelogenous leukemia cell line. Cells were maintained in medium with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA).
624 %
Determination of the serum concentration of lentinan in cancer patients
J
60
After the intravenous infusion of lentinan (1 mg), samples were collected and the serum lentinan concentration was determined using the Toxicolor test (Seikagaku Kogyo, Tokyo, Japan) [12].
Results /.0
Optimal dose of lentinan and optimal incubation period for the induction of killer cells
x
{.D
o~
20
0
f
P
Lentinan (-)
Lentinan (,)
Fig.1. Cytotoxicity of PBMC against K562 cells after stimulation with lentinan in vitro
After 2 days of culture of PBMC with lentinan 12,000 ng.m1-1, cytotoxicity was not increased (Fig. 1). However, after 4 days of culture, an increase in cytotoxicity was observed (P < 0.01), and the concentration of lentinan that augmented cytotoxic activity was always less than 1,000 ng. ml - ~(Fig. 2). After 8 days of culture, the cytotoxicity was 11.9% of the maximal increase (P < 0.01; Fig. 3), and the optimal lentinan concentration was below 1,000 ng. ml- 1. The dose titration of lentinan for cytotoxic activity is shown in Fig. 4. The optimal concentration of lentinan showed 2 peaks, one from 5 to 25 ng. ml-1 and 1,000 ng. ml- 1for the 8-day culture (P < 0.01, Fig. 4).
Induction of cytotoxicmacrophages by lentinan Cytotoxic assay A 4-h 5~Cr-release assay was performed for killer cells and a 16-h ~25Irelease assay was performed for cytotoxic macrophages. The target cells were labelled for 1 h at 37 °C with 100 gCi Na251CrO4 or with 125Ideoxyuridine for I h at 37 °C, and then washed three-times with medium containing 10% FCS. Then, 100 ~tl 51Cr-labeled tumour cells (1 x 10S/ml) was added in triplicate to 100 ~tl effector cells (the effector-to-target ratio was fixed at 15:1) in round-bottomed microtitre plates (Corning No.25850). After a 4-h incubation for killer cells, and a 16-h incubation for macrophages, both at 37 °C, the supernatants were harvested and the radioactivity was counted with a gamma counter. Spontaneous release was determined by the incubation of labelled target cells in complete medium alone, and did not exceed 10% of the maximum release following the addition of 1 M HC1. The percentage specific cytotoxicity was calculated as; (all 51Cr or ~25Ivalues were in cpm): test release-spontaneous release x 100 maximum release-spontaneous release
The cytotoxicity of splenic macrophages for K562 cells was increased from 0.5% to 51.5% (mean 18.8% ) by lentinan (P < 0.05; Fig. 5), and the optimal concentration of lentinan was 25 to 250 ng. ml- 1, similar to that for PBMC (Fig. 6). % P
