Biochimica et Biophysica Acta, 1138(1992) 157-161
157
© 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00
BBADIS 61115
Stimulation of arachidonic acid metabolism by a streptococcal preparation (OK-432) in rat peritoneal macrophages Masako Watanabe 1, Yoshiyuki Shishido 1, Noriyasu Hirasawa l, Suetsugu Mue 2, Hirota Fujiki 3 and Kazuo Ohuchi : Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku Unit'ersity, Miyagi (Japan), 2 College of Medical Sciences, Tohoku Unit,ersity, Miyagi (Japan) and ~ Cancer Chemoprecention Di~:ision, National Cancer Center Research Institute, Tokyo (Japan)
(Received 20 June 1991)
Key words: OK-432;Prostaglandin E2; Arachidonicacid; Phagocytosis;CytochalasinB; (Rat peritoneal macrophage) A streptococcal preparation OK-432 is reported to be an immunopotentiator and a potent antitumor agent. In order to elucidate the mechanism of biologic action, effects of OK-432 on arachidonic acid metabolism in rat peritoneal macrophages were investigated. Prostaglandin E 2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were found to be stimulated by OK-432 in a concentration-dependent manner (5 to 80 # g / m l ) . Heat-treatment of OK-432 further stimulated its effects. These stimulative effects on arachidonic acid metabolism by OK-432 were not observed in MDCK cells that have no phagocytotic activity. Furthermore, cytochalasin B treatment completely suppressed the stimulative effects induced by OK-432 in macrophages. These results strongly indicate that the stimulative effects by OK-432 on arachidonic acid metabolism are dependent on phagocytosis of OK-432 particles. Significance of stimulation of arachidonic acid metabolism in macrophages by OK-432 for its biological effects is discussed.
Introduction
OK-432, is a heat- and penicillin-treated, lyophylized preparation of the Su strain of Streptococcus pyogenes A3 [1], and is reported to activate macrophages [2], natural killer cells [3] and T lymphocytes [4] through various cytokines such as interferon [5], interleukin-1 and -2 [6] and tumor necrosis factor [7]. OK-432 has been proved to augment host-defence mechanisms against tumors in animals [8,9] and human [10-12]. Thus, OK-432 has been used for clinical cancer-immunotherapy in Japan. However, the antitumor mechanism of OK-432 has not been fully understood. The most probable mechanism might be an activation of cytotoxic host cells, especially macrophages [13,14]. In fact, it is reported that mouse peritoneal macrophages become cytotoxic for tumor cells after intraperitoneal injection of OK-432 [2]. Macrophage functions such as motility, adhesiveness and interleukin-1 production are
also activated by the intraperitoneal injection of OK432 [2]. In this report, effects of OK-432 on arachidonic acid metabolism in rat peritoneal macrophages in culture were investigated in order to gain further insight into the mechanism of biological actions of OK-432 in vivo. Materials and Methods Preparation o f rat peritoneal macrophages
A solution containing soluble starch (Wako Pure Chemical, Tokyo, Japan) and bacto peptone (Difco Laboratories, Detroit, MI, U.S.A.) (5% each) was injected into male rats (the Sprague-Dawley strain, specific pathogen-free, Charles River, Japan) intraperitoneally at a dose of 5 ml per 100 g body weight. 4 days after the injection, the rats were killed by cutting the carotid artery under diethyl ether anesthesia and peritoneal cells were harvested [15]. Macrophage culture
Correspondence: M. Watanabe, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980, Japan.
The cells were suspended in Eagle's minimum essential medium (Nissui, Tokyo, Japan) supplemented with 10% (v/v) calf bovine serum (Grand Island Bio-
158 chemical, Grand Island, NY, U.S.A.), penicillin G potassium (18 # g / m l ) and streptomycin sulfate (50 # g / m l ) (Meiji Seika, Tokyo, Japan). The macrophages were seeded at 6 . 1 0 6 cells in 4 ml of the medium per 60 mm plastic tissue culture dish (Iwaki Glass, Tokyo, Japan) and incubated for 2 h at 37°C. After the incubation, the dishes were washed three times with the medium to wash out non-adherent cells [15].
Measurement of radioacticity from [ 3H/arachidonic acid-labeled macrophages The adherent cells were further incubated in 4 ml of the medium containing 37 KBq of [3H]arachidonic acid (2.26 T B q / m m o l , New England Nuclear, Boston, MA, U.S.A.) for 20 h at 37°C in order to label the cellular lipids [15]. To remove free [3H]arachidonic acid, the adherent cells were washed three times with the medium and then incubated in 4 ml of the medium containing 10% ( v / v ) calf bovine serum and various concentrations of drugs examined. At the appropriate times of incubation, 100 /xl of the medium was withdrawn and counted for the released radioactivity [15].
Radioimmunoassay of prostaglandin
E2
Prostaglandin E 2 levels in the medium were radioimmunoassayed [15] after incubating the non-labeled macrophages (6.106 cells) in 4 ml of the medium supplemented with 10% ( v / v ) calf bovine serum (Grand Island Biochemical) and containing various concentrations of drugs examined. Prostaglandin E 2 antiserum was purchased from Seragen (Boston, MA, U.S.A.).
Chemicals OK-432, a streptococcal antitumor agent Picibanil, was produced by Chugai Pharmaceutical (Tokyo, Japan), by incubating low virulence, Su type IIl, group A Streptococcus pyogenes in Bernheimer's basal medium with penicillin G, followed by lyophilization of the incubation mixture [1]. OK-432 thus obtained was suspended in Eagle's minimum essential medium (1 m g / m l ) , and an aliquot of the suspension was added into the incubation medium for macrophages. Heattreated OK-432 was obtained by autoclaving (120°C for 20 min) the suspension of normal OK-432 (1 m g / m l ) . Cytochalasin B (Sigma, St. Louis, MI, U.S.A.) was dissolved in dimethylsulfoxide and an aliquot of the solution was added into the medium. Final concentration of dimethylsulfoxide was adjusted to (/.2% (v/v). Control medium contained the same amount of the vehicle. Indomethacin (Sigma) was dissolved in ethanol and added into the incubation medium. Final concentration of ethanol was adjusted to 0.2% (v/v). Control medium contained the same amount of the vehicle.
Statistical analysis Results were analyzed for statistical significance by Student's t test for unpaired observations. Results
Release of radioactivity from [3H]arachidonic acidlabeled macrophages was stimulated by OK-432 treatment in a concentration-dependent manner (Fig. 1). At concentrations of 20 and 80 /~g/ml, significant in-
MDCK cell culture Madin-Darby canine kidney cells ( M D C K cells) were purchased from Dainippon Pharm. (Osaka, Japan). After 5 passages in the medium used for macrophage culture, they were seeded at 2 • 105 cells in 4 ml of the medium per 60 mm plastic culture dish (Iwaki Glass) for 20 h at 37°C. The cells were then washed 3 times with the medium and incubated at 37°C for 4 h in 4 ml of the medium containing various concentrations of drugs examined. Prostagalndin E 2 levels in the medium were radioimmunoassayed as described above. For measurement of release of radioactivity, 2.105 cells were incubated in 4 ml of the medium containing 37 KBq of [3H]arachidonic acid (2.26 T B q / m m o l , New England Nuclear) for 20 h at 37°C in order to label cellular lipids. The cells were then washed three times with the medium and further incubated in 4 ml of the medium supplemented with 10% calf bovine serum (Grand Island Biochemical) and containing various concentrations of drugs. At the appropriate times of incubation, 100 Izl of the medium was withdrawn and counted for the released radioactivity.
>"
4
80
>
~5
x
"O
~.
20
~5 2 5 0
ct"
0
1
2 Incubotion
4
6
Time (h)
Fig. 1. Time-course of release of radioactivity from [3H]arachidonic acid-labeled macrophages. [3H]Arachidonic acid-labeled macrophages (6.106 cells) were incubated at 37°C in 4 ml of the medium containing indicated concentrations of OK-432 (~tg/ml). Total radioactivity in 4 ml of the medium was measured. Values are the means _+S.E. from four dishes. Each S.E. was too small to depict. Statistical significance: OK-432 5 tzg/ml vs. control, P < 0.01 at 4 h, P < 0.001 at 6 h; OK-432 20 or 80/zg/ml vs. control. P < 0.001 at 1, 2, 4 and 6 h.
159
O
~2
E o, C
10
i_;~ 2
~, 20
× C
re"
t-
5
O
g,
10
O 13K
A-
0
0K-432
0 0
5
0K-432
20 ( pg/ml
80 )
Fig. 2. Effects of various concentrations of OK-432 on prostaglandin E 2 production. Macrophages (6.106 cells) were incubated at 37°C in 4 ml of the medium containing indicated concentrations ( / x g / m l ) of OK-432. Prostaglandin E 2 levels in the medium 4 h after incubation were radioimmunoassayed. Values are the m e a n s + S . E , from four dishes. Statistical significance: OK-432 5 # g / m l vs. control, P < 0.01; OK-432 20 or 8 0 / x g / m l vs. control, P < 0.001.
creases were induced 1 to 6 h after incubation. However, at a concentration of 5 p.g/ml, significant increases were induced 4 and 6 h after incubation. A concentration-dependent increase in prostaglandin E 2 production by OK-432 treatment also was observed when measured 4 h after incubation at concentrations of 5 to 80/.~g/ml (Fig. 2). Release of radioactivity and prostaglandin E 2 production in M D C K cells, non-phagocytic cells, measured 6 h after incubation were not stimulated by OK-432 treatment at concentrations up to 80 ~ g / m l (data not shown). OK-432-induced release of radioactivity in rat peritoneal macrophages at a concentration of 20 ~zg/ml was inhibited by cytochalasin B in a concentration-dependent manner when measured 4 h after incubation (Fig. 3). At a concentration of 10 /~g/ml, cytochalasin B suppressed the OK-432-induced release of radioactivity almost to the same level of spontaneous release (Fig. 3). Spontaneous release of radioactivity was not affected by cytochalasin B treatment at concentrations up to 10 i z g / m l when measured 4 h after incubation (data not shown). Stimulation of prostaglandin E 2 production induced by OK-432 at a concentration of 20 t~g/ml also was inhibited by cytochalasin B in a concentration-dependent manner when measured 4 h after incubation (Fig. 3). At a concentration of 10 p.g/ml, OK-432-induced stimulation of prostaglandin E 2 production was completely suppressed (Fig. 3). Release of radioactivity from [3H]arachidonic acidlabeled macrophages was much more stimulated when the macrophages were incubated in the medium containing heat-treated OK-432 than in the medium containing normal OK-432 each at a concentration of 20 i z g / m l (Fig. 4). Prostaglandin E 2 levels in the medium both 2 and 4 h after incubation also were much more
Cytocholasin B
[3.
0
0 0
20 0
0.1
1
10
Fig. 3. Effects of cytochalasin B on the release of radioactivity and prostaglandin E 2 production stimulated by OK-432. [3H]Arachidonic acid-labeled macrophages or non-labeled macrophages (6' 106 cells) were incubated at 37°C in 4 ml of the medium containing OK-432 (20 p , g / m l ) and indicated concentrations of cytochalasin B (/~g/ml). Released radioactivity and prostaglandin E 2 levels were measured 4 h after incubation. Values are the m e a n s + S . E , from four dishes. Hatched columns and closed columns represent release of radioactivity and prostaglandin E 2 levels, respectively. Statistical significance: OK-432 20+cytochalasin B 1 or 10 vs. OK-432 20+cytochalasin B 0, P < 0.001; OK-432 20+cytochalasin B 0, 0.1 or 1 vs. OK-432 0 plus cytochalasin B 0, P < 0.001, both in the release of radioactivity and prostaglandin E 2 production.
increased by incubating the cells in the medium containing heat-treated OK-432 than in the medium containing normal OK-432 each at a concentration of 20 /~g/ml (Fig. 4). 4 h after incubation, both the release of radioactivity and prostaglandin E 2 production stimulated by treatment with heat-treated OK-432 at a concentration of 20 ~ g / m l were suppressed to the levels of n o n - t r e a t e d group by incubating the macrophages in the presence of cytochalasin B at a concentration of 10 I z g / m l (Fig. 5). When the macrophages were incubated in the medium containing
20
E o~ C
10
~ o
£ O
~X O~
0 0K-432
.c
.MI 0
20N 2h
20H
o 0 0
20N
20H
4h
Fig. 4. Effects of normal OK-432 and heat-treated OK-432 on the release of radioactivity and prostaglandin E 2 production. [3H]Arachidonic acid-labeled macrophages or non-labeled macrophages (6.106 cells) were incubated at 37°C for indicated period in 4 ml of the medium containing 20 p , g / m l of normal OK-432 (N) or heattreated OK-432 (H). Hatched columns and closed columns represent release of radioactivity and prostaglandin E 2 levels, respectively. Values are the m e a n s + S.E. from four dishes. Statistical significance: At 2 and 4 h, OK-432 20 vs. 0, P < 0.001; OK-432 20N vs. OK-432 20H, P < 0.001, both in the release of radioactivity and prostaglandin E 2 production.
160 fi 20 u 'o rY
~ ~
c
~7 c ,g
o
10
2
o o L n
0 0K-432 Cytocholosin B
20H
0
0
10
Fig. 5. Effects of cytochalsin B on the release of radioactivity and prostaglandin E 2 production stimulated by heat-treated OK-432. [3H]Arachidonic acid-labeled macrophages or non-labeled macrophages (6.1() ~' cells) were incubated at 37°C for 4 h in 4 ml of the medium containing 20 /~g/ml of heat-treated OK-432 (H) with or without cytochalasin B (ll) /zg/ml). Hatched columns and closed columns represent release of radioactivity and prostaglandin E~ levels, respectively. Values are the means_+S.E, from four dishes. Statistical significance: In the absence of cytochalsin B: OK-432 20H vs. 0, P < 0,001: in the presence of OK-432: cytochalasin B 0 vs. 10, P < 0.(101, both in the release of radioactivity and prostaglandin E 2 production.
indomethacin at a concentration of 1 0 / z g / m l , prostaglandin E 2 production 4 h after incubation was almost completely suppressed both in the non-treated group and OK-432 treated group (data not shown). Discussion
Present investigations revealed that OK-432 has an activity to stimulate arachidonic acid metabolism in rat peritoneal macrophages. Rat peritoneal macrophages from the Sprauge-Dawley strain synthesize a small amount of lipoxygenase products, such as leukotorienes B 4, C 4, D 4, and E 4 by various stimulations [16,17], but synthesize cyclooxygenase products such as prostaglandin E 2 and 6-keto-prostaglandin FI~~ in a large quantity [16,17]. In the present work, by HPLC analysis [18], effects of OK-432 on the production of such leukotrienes in rat peritoneal macrophages were examined. However, OK-432 at concentrations up to 80 /zg/ml failed to stimulate the production of these leukotrienes (data not shown). Therefore, prostaglandin E 2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were measured separately as indices of stimulation of arachidonic acid metabolism. In consequence, OK-432 at concentrations of 5 to 80 t~g/ml has been proved to stimulate both prostaglandin E 2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages (Figs. 1 and 2). Microscopic observations revealed that OK-432 is not dissolved but is suspended in the medium even at a concentration of 5 ~ g / m l . Furthermore, the macrophages phagocytozed OK-432 particles during the incubation period. Consequently,
OK-432-induced stimulation of arachidonic acid metabolism seemed to be closely related with phagocytosis of OK-432 particles. Indeed, OK-432 failed to stimulate arachidonic acid metabolism in MDCK cells that do not phagocytoze OK-432 particles (data not shown). In addition, it was clearly demonstrated that OK-432-induced stimulation of arachidonic acid metabolism is suppressed completely by treatment with cytochalasin B, an inhibitor of phagocytosis [19], at a concentration of 10 ~ g / m l (Fig. 3). At this concentration of cytochalasin B, phagocytosis of OK-432 particles also was completely inhibited as revealed by microscopic observations (data not shown). These results strongly indicate that the OK-432-induced stimulation of arachidonic acid metabolism in macrophages is induced by phagocytosis of foreign bodies, OK-432 particles. When OK-432 was heat-treated (120°C for 20 min), stimulative effects on arachidonic acid metabolism was further increased than by non-treated OK-432 (Fig. 4). This increased activity was also inhibited completely by cytochalasin B treatment (Fig. 5), suggesting that the increase in arachidonic acid metabolism by heat-treated OK-432 is aslo induced by phagocytosis of OK-432 particles. Microscopic observations revealed that nontreated OK-432 particles easily form aggregates in the incubation medium, however, after heat treatment OK432 particles tend to disaggregate in the medium. Consequently, increased activity by heat-treated OK-432 to stimulate arachidonic acid metabolism (Fig. 4) might be due to the qualitative changes of OK-432, viz. the particles of heat-treated OK-432 remained to be more phagocytozable forms in the medium than the nontreated OK-432, which easily form aggregates that would be less phagocytozable than free OK-432 particles. Kawaguchi et al. [2] reported that mouse peritoneal macrophages collected after intraperitoneal injection of OK-432 showed increased motility, adhesiveness, interleukin-1 production, and inhibition of tumor cell growth in vitro. However, when heat-treated OK-432 (120°C for 20 min) was injected, the peritoneal macrophages showed no activity to induce inhibition of tumor cell growth and production of interleukin-1, but showed the increased motility and adhesiveness [2]. Consequently, stimulation of arachidonic acid metabolism by OK-432 might be related with increased motility and adhesiveness of macrophages but not with induction of inhibition of tumor cells growth and interleukin-1 production. However, the activation of macrophages as reflected by stimulation of arachidonic acid metabolism by OK-432 might play some significant roles in immunopotentiation that leads to antitumor effects demonstrated experimentally [4,13,20] and clinically [10,21-23]. Although the present work revealed that OK-432 has an activity to stimulate arachidonic
161 acid metabolism in macrophages, further studies are necessary to clarify mechanism of antitumor action of OK-432 in vivo.
Acknowledgement This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan. References l Okamoto, H., Shoin, S., Koshimura, S. and Shimizu, R. (1967) Jpn. J. Microbiol. 11,323-336. 2 Kawaguchi, T., Suematsu, M., Masuda-Koizumi, H., Mitsui, H., Suzuki, S., Matsuno, T., Ogawa, H. and Nomoto, K. (1983) Immunopharmacology 6, 177-189. 3 Uchida, A. and Micksche, M. (1983) Int. J. Cancer 31, 1-5. 4 Hojo, H. and Hashimoto, Y. (1981) Gann 72, 692-699. 5 Saito, M., Ebina, T., Koi, M., Yamaguchi, T., Kawade, Y. and Ishida, N. (1982) Cell. Immunol. 68, 187-192. 6 lchimura, O., Suzuki, S., Saito, M., Sugawara, Y. and Ishida, N. (1985) Int. J. lmmunopharmacol. 7, 263-270. 7 Yamamoto, A., Nagamuta, M., Usami, Y., Sugawara, N., Watanabe, Y., Niitsu, Y. and Urushizaki, I. (1986) Immunopharmacology 11, 79-86. 8 Sakurai, Y., Tsukagoshi, S., Satoh, H., Akiba, T., Suzuki, S. and Takagi, Y. (1972) Cancer Chemother. Rep. 56, 9-17.
9 Yamagishi, H., Pellis, N.R. and Kahan, B.D. (1980) Cancer Immunol. Immunother. 9, 63-67. 10 Uchida, A., and Hoshino, T. (1980) Cancer 45, 476-483. 11 Torisu, M., Katano, M., Kimura, H., Itoh, H. and Takesue, M. (1983) Surgery 93, 357-364. 12 Watanabe, Y. and lwa, T. (1984) Cancer 53, 248-253. 13 Ishii, Y., Yamamoto, H., Toh, K. and Kikuchi, K. (1976) Gann 67, 115-119. 14 Torikai, T., ]toh, O., Satoh, M. and Osawa, T. (1981)) Gann 71, 52-59. 15 Watanabe, M., Tamura, T., Ohashi, M., Hirasawa, N., Ozeki, T., Tsurufuji, S., Fujiki, H. and Ohuchi, K. (1990) Biochim. Biophys. Acta 1/)47, 141-147. 16 Ohuchi, K., Sugawara, T., Watanabe, M., Hirasawa, N., Tsurufuji, S., Fujiki, H., Sugimura, T. and Christensen, S.B. (1987) J. Cancer Res. Clin. Oncol. 113, 319-324. 17 Ohuchi, K., Watanabe, M., Hirasawa, N., Tsurufuji, S., Ozeki, T. and Fujiki, H. (1988) Biochim. Biophys. Acta 971, 85-91. 18 Ohuchi, K., Takahashi, C., Watanabe, M., Hirasawa, N., Suzuki, Y., Kudo, M., Konishi, T. and Tsurufuji, S. (1988) J. Clin. Lab. Immunol. 27, 171 178. 19 Zigmond, S.H. and Hirsch, J.G. (1972) Exp. Cell Res. 73,383-393. 20 Aoki, T., Kvedar, J.P., Hollis, V.W. Jr. and Bushar, G.S. (1976) J. Natl. Cancer Inst. 56, 687-690. 21 Kurokawa, T., Hanori, T. and Furue, H. (1972) Cancer Chemother. Rep. 56, 211-220. 22 Kimura, I., Ohnoshi, T., Yasuhara, S., Sugiyama, M., Urabe, Y., Fujii, M. and Machida, K. (1976) Cancer 37, 2201-221)3. 23 Tanaka, R., Sekiguchi, K., Suzuki, Y., Sobue, H. and Ueki, K. (1980) Cancer 46, 1688-1694.
157
© 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00
BBADIS 61115
Stimulation of arachidonic acid metabolism by a streptococcal preparation (OK-432) in rat peritoneal macrophages Masako Watanabe 1, Yoshiyuki Shishido 1, Noriyasu Hirasawa l, Suetsugu Mue 2, Hirota Fujiki 3 and Kazuo Ohuchi : Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku Unit'ersity, Miyagi (Japan), 2 College of Medical Sciences, Tohoku Unit,ersity, Miyagi (Japan) and ~ Cancer Chemoprecention Di~:ision, National Cancer Center Research Institute, Tokyo (Japan)
(Received 20 June 1991)
Key words: OK-432;Prostaglandin E2; Arachidonicacid; Phagocytosis;CytochalasinB; (Rat peritoneal macrophage) A streptococcal preparation OK-432 is reported to be an immunopotentiator and a potent antitumor agent. In order to elucidate the mechanism of biologic action, effects of OK-432 on arachidonic acid metabolism in rat peritoneal macrophages were investigated. Prostaglandin E 2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were found to be stimulated by OK-432 in a concentration-dependent manner (5 to 80 # g / m l ) . Heat-treatment of OK-432 further stimulated its effects. These stimulative effects on arachidonic acid metabolism by OK-432 were not observed in MDCK cells that have no phagocytotic activity. Furthermore, cytochalasin B treatment completely suppressed the stimulative effects induced by OK-432 in macrophages. These results strongly indicate that the stimulative effects by OK-432 on arachidonic acid metabolism are dependent on phagocytosis of OK-432 particles. Significance of stimulation of arachidonic acid metabolism in macrophages by OK-432 for its biological effects is discussed.
Introduction
OK-432, is a heat- and penicillin-treated, lyophylized preparation of the Su strain of Streptococcus pyogenes A3 [1], and is reported to activate macrophages [2], natural killer cells [3] and T lymphocytes [4] through various cytokines such as interferon [5], interleukin-1 and -2 [6] and tumor necrosis factor [7]. OK-432 has been proved to augment host-defence mechanisms against tumors in animals [8,9] and human [10-12]. Thus, OK-432 has been used for clinical cancer-immunotherapy in Japan. However, the antitumor mechanism of OK-432 has not been fully understood. The most probable mechanism might be an activation of cytotoxic host cells, especially macrophages [13,14]. In fact, it is reported that mouse peritoneal macrophages become cytotoxic for tumor cells after intraperitoneal injection of OK-432 [2]. Macrophage functions such as motility, adhesiveness and interleukin-1 production are
also activated by the intraperitoneal injection of OK432 [2]. In this report, effects of OK-432 on arachidonic acid metabolism in rat peritoneal macrophages in culture were investigated in order to gain further insight into the mechanism of biological actions of OK-432 in vivo. Materials and Methods Preparation o f rat peritoneal macrophages
A solution containing soluble starch (Wako Pure Chemical, Tokyo, Japan) and bacto peptone (Difco Laboratories, Detroit, MI, U.S.A.) (5% each) was injected into male rats (the Sprague-Dawley strain, specific pathogen-free, Charles River, Japan) intraperitoneally at a dose of 5 ml per 100 g body weight. 4 days after the injection, the rats were killed by cutting the carotid artery under diethyl ether anesthesia and peritoneal cells were harvested [15]. Macrophage culture
Correspondence: M. Watanabe, Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980, Japan.
The cells were suspended in Eagle's minimum essential medium (Nissui, Tokyo, Japan) supplemented with 10% (v/v) calf bovine serum (Grand Island Bio-
158 chemical, Grand Island, NY, U.S.A.), penicillin G potassium (18 # g / m l ) and streptomycin sulfate (50 # g / m l ) (Meiji Seika, Tokyo, Japan). The macrophages were seeded at 6 . 1 0 6 cells in 4 ml of the medium per 60 mm plastic tissue culture dish (Iwaki Glass, Tokyo, Japan) and incubated for 2 h at 37°C. After the incubation, the dishes were washed three times with the medium to wash out non-adherent cells [15].
Measurement of radioacticity from [ 3H/arachidonic acid-labeled macrophages The adherent cells were further incubated in 4 ml of the medium containing 37 KBq of [3H]arachidonic acid (2.26 T B q / m m o l , New England Nuclear, Boston, MA, U.S.A.) for 20 h at 37°C in order to label the cellular lipids [15]. To remove free [3H]arachidonic acid, the adherent cells were washed three times with the medium and then incubated in 4 ml of the medium containing 10% ( v / v ) calf bovine serum and various concentrations of drugs examined. At the appropriate times of incubation, 100 /xl of the medium was withdrawn and counted for the released radioactivity [15].
Radioimmunoassay of prostaglandin
E2
Prostaglandin E 2 levels in the medium were radioimmunoassayed [15] after incubating the non-labeled macrophages (6.106 cells) in 4 ml of the medium supplemented with 10% ( v / v ) calf bovine serum (Grand Island Biochemical) and containing various concentrations of drugs examined. Prostaglandin E 2 antiserum was purchased from Seragen (Boston, MA, U.S.A.).
Chemicals OK-432, a streptococcal antitumor agent Picibanil, was produced by Chugai Pharmaceutical (Tokyo, Japan), by incubating low virulence, Su type IIl, group A Streptococcus pyogenes in Bernheimer's basal medium with penicillin G, followed by lyophilization of the incubation mixture [1]. OK-432 thus obtained was suspended in Eagle's minimum essential medium (1 m g / m l ) , and an aliquot of the suspension was added into the incubation medium for macrophages. Heattreated OK-432 was obtained by autoclaving (120°C for 20 min) the suspension of normal OK-432 (1 m g / m l ) . Cytochalasin B (Sigma, St. Louis, MI, U.S.A.) was dissolved in dimethylsulfoxide and an aliquot of the solution was added into the medium. Final concentration of dimethylsulfoxide was adjusted to (/.2% (v/v). Control medium contained the same amount of the vehicle. Indomethacin (Sigma) was dissolved in ethanol and added into the incubation medium. Final concentration of ethanol was adjusted to 0.2% (v/v). Control medium contained the same amount of the vehicle.
Statistical analysis Results were analyzed for statistical significance by Student's t test for unpaired observations. Results
Release of radioactivity from [3H]arachidonic acidlabeled macrophages was stimulated by OK-432 treatment in a concentration-dependent manner (Fig. 1). At concentrations of 20 and 80 /~g/ml, significant in-
MDCK cell culture Madin-Darby canine kidney cells ( M D C K cells) were purchased from Dainippon Pharm. (Osaka, Japan). After 5 passages in the medium used for macrophage culture, they were seeded at 2 • 105 cells in 4 ml of the medium per 60 mm plastic culture dish (Iwaki Glass) for 20 h at 37°C. The cells were then washed 3 times with the medium and incubated at 37°C for 4 h in 4 ml of the medium containing various concentrations of drugs examined. Prostagalndin E 2 levels in the medium were radioimmunoassayed as described above. For measurement of release of radioactivity, 2.105 cells were incubated in 4 ml of the medium containing 37 KBq of [3H]arachidonic acid (2.26 T B q / m m o l , New England Nuclear) for 20 h at 37°C in order to label cellular lipids. The cells were then washed three times with the medium and further incubated in 4 ml of the medium supplemented with 10% calf bovine serum (Grand Island Biochemical) and containing various concentrations of drugs. At the appropriate times of incubation, 100 Izl of the medium was withdrawn and counted for the released radioactivity.
>"
4
80
>
~5
x
"O
~.
20
~5 2 5 0
ct"
0
1
2 Incubotion
4
6
Time (h)
Fig. 1. Time-course of release of radioactivity from [3H]arachidonic acid-labeled macrophages. [3H]Arachidonic acid-labeled macrophages (6.106 cells) were incubated at 37°C in 4 ml of the medium containing indicated concentrations of OK-432 (~tg/ml). Total radioactivity in 4 ml of the medium was measured. Values are the means _+S.E. from four dishes. Each S.E. was too small to depict. Statistical significance: OK-432 5 tzg/ml vs. control, P < 0.01 at 4 h, P < 0.001 at 6 h; OK-432 20 or 80/zg/ml vs. control. P < 0.001 at 1, 2, 4 and 6 h.
159
O
~2
E o, C
10
i_;~ 2
~, 20
× C
re"
t-
5
O
g,
10
O 13K
A-
0
0K-432
0 0
5
0K-432
20 ( pg/ml
80 )
Fig. 2. Effects of various concentrations of OK-432 on prostaglandin E 2 production. Macrophages (6.106 cells) were incubated at 37°C in 4 ml of the medium containing indicated concentrations ( / x g / m l ) of OK-432. Prostaglandin E 2 levels in the medium 4 h after incubation were radioimmunoassayed. Values are the m e a n s + S . E , from four dishes. Statistical significance: OK-432 5 # g / m l vs. control, P < 0.01; OK-432 20 or 8 0 / x g / m l vs. control, P < 0.001.
creases were induced 1 to 6 h after incubation. However, at a concentration of 5 p.g/ml, significant increases were induced 4 and 6 h after incubation. A concentration-dependent increase in prostaglandin E 2 production by OK-432 treatment also was observed when measured 4 h after incubation at concentrations of 5 to 80/.~g/ml (Fig. 2). Release of radioactivity and prostaglandin E 2 production in M D C K cells, non-phagocytic cells, measured 6 h after incubation were not stimulated by OK-432 treatment at concentrations up to 80 ~ g / m l (data not shown). OK-432-induced release of radioactivity in rat peritoneal macrophages at a concentration of 20 ~zg/ml was inhibited by cytochalasin B in a concentration-dependent manner when measured 4 h after incubation (Fig. 3). At a concentration of 10 /~g/ml, cytochalasin B suppressed the OK-432-induced release of radioactivity almost to the same level of spontaneous release (Fig. 3). Spontaneous release of radioactivity was not affected by cytochalasin B treatment at concentrations up to 10 i z g / m l when measured 4 h after incubation (data not shown). Stimulation of prostaglandin E 2 production induced by OK-432 at a concentration of 20 t~g/ml also was inhibited by cytochalasin B in a concentration-dependent manner when measured 4 h after incubation (Fig. 3). At a concentration of 10 p.g/ml, OK-432-induced stimulation of prostaglandin E 2 production was completely suppressed (Fig. 3). Release of radioactivity from [3H]arachidonic acidlabeled macrophages was much more stimulated when the macrophages were incubated in the medium containing heat-treated OK-432 than in the medium containing normal OK-432 each at a concentration of 20 i z g / m l (Fig. 4). Prostaglandin E 2 levels in the medium both 2 and 4 h after incubation also were much more
Cytocholasin B
[3.
0
0 0
20 0
0.1
1
10
Fig. 3. Effects of cytochalasin B on the release of radioactivity and prostaglandin E 2 production stimulated by OK-432. [3H]Arachidonic acid-labeled macrophages or non-labeled macrophages (6' 106 cells) were incubated at 37°C in 4 ml of the medium containing OK-432 (20 p , g / m l ) and indicated concentrations of cytochalasin B (/~g/ml). Released radioactivity and prostaglandin E 2 levels were measured 4 h after incubation. Values are the m e a n s + S . E , from four dishes. Hatched columns and closed columns represent release of radioactivity and prostaglandin E 2 levels, respectively. Statistical significance: OK-432 20+cytochalasin B 1 or 10 vs. OK-432 20+cytochalasin B 0, P < 0.001; OK-432 20+cytochalasin B 0, 0.1 or 1 vs. OK-432 0 plus cytochalasin B 0, P < 0.001, both in the release of radioactivity and prostaglandin E 2 production.
increased by incubating the cells in the medium containing heat-treated OK-432 than in the medium containing normal OK-432 each at a concentration of 20 /~g/ml (Fig. 4). 4 h after incubation, both the release of radioactivity and prostaglandin E 2 production stimulated by treatment with heat-treated OK-432 at a concentration of 20 ~ g / m l were suppressed to the levels of n o n - t r e a t e d group by incubating the macrophages in the presence of cytochalasin B at a concentration of 10 I z g / m l (Fig. 5). When the macrophages were incubated in the medium containing
20
E o~ C
10
~ o
£ O
~X O~
0 0K-432
.c
.MI 0
20N 2h
20H
o 0 0
20N
20H
4h
Fig. 4. Effects of normal OK-432 and heat-treated OK-432 on the release of radioactivity and prostaglandin E 2 production. [3H]Arachidonic acid-labeled macrophages or non-labeled macrophages (6.106 cells) were incubated at 37°C for indicated period in 4 ml of the medium containing 20 p , g / m l of normal OK-432 (N) or heattreated OK-432 (H). Hatched columns and closed columns represent release of radioactivity and prostaglandin E 2 levels, respectively. Values are the m e a n s + S.E. from four dishes. Statistical significance: At 2 and 4 h, OK-432 20 vs. 0, P < 0.001; OK-432 20N vs. OK-432 20H, P < 0.001, both in the release of radioactivity and prostaglandin E 2 production.
160 fi 20 u 'o rY
~ ~
c
~7 c ,g
o
10
2
o o L n
0 0K-432 Cytocholosin B
20H
0
0
10
Fig. 5. Effects of cytochalsin B on the release of radioactivity and prostaglandin E 2 production stimulated by heat-treated OK-432. [3H]Arachidonic acid-labeled macrophages or non-labeled macrophages (6.1() ~' cells) were incubated at 37°C for 4 h in 4 ml of the medium containing 20 /~g/ml of heat-treated OK-432 (H) with or without cytochalasin B (ll) /zg/ml). Hatched columns and closed columns represent release of radioactivity and prostaglandin E~ levels, respectively. Values are the means_+S.E, from four dishes. Statistical significance: In the absence of cytochalsin B: OK-432 20H vs. 0, P < 0,001: in the presence of OK-432: cytochalasin B 0 vs. 10, P < 0.(101, both in the release of radioactivity and prostaglandin E 2 production.
indomethacin at a concentration of 1 0 / z g / m l , prostaglandin E 2 production 4 h after incubation was almost completely suppressed both in the non-treated group and OK-432 treated group (data not shown). Discussion
Present investigations revealed that OK-432 has an activity to stimulate arachidonic acid metabolism in rat peritoneal macrophages. Rat peritoneal macrophages from the Sprauge-Dawley strain synthesize a small amount of lipoxygenase products, such as leukotorienes B 4, C 4, D 4, and E 4 by various stimulations [16,17], but synthesize cyclooxygenase products such as prostaglandin E 2 and 6-keto-prostaglandin FI~~ in a large quantity [16,17]. In the present work, by HPLC analysis [18], effects of OK-432 on the production of such leukotrienes in rat peritoneal macrophages were examined. However, OK-432 at concentrations up to 80 /zg/ml failed to stimulate the production of these leukotrienes (data not shown). Therefore, prostaglandin E 2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages were measured separately as indices of stimulation of arachidonic acid metabolism. In consequence, OK-432 at concentrations of 5 to 80 t~g/ml has been proved to stimulate both prostaglandin E 2 production and release of radioactivity from [3H]arachidonic acid-labeled macrophages (Figs. 1 and 2). Microscopic observations revealed that OK-432 is not dissolved but is suspended in the medium even at a concentration of 5 ~ g / m l . Furthermore, the macrophages phagocytozed OK-432 particles during the incubation period. Consequently,
OK-432-induced stimulation of arachidonic acid metabolism seemed to be closely related with phagocytosis of OK-432 particles. Indeed, OK-432 failed to stimulate arachidonic acid metabolism in MDCK cells that do not phagocytoze OK-432 particles (data not shown). In addition, it was clearly demonstrated that OK-432-induced stimulation of arachidonic acid metabolism is suppressed completely by treatment with cytochalasin B, an inhibitor of phagocytosis [19], at a concentration of 10 ~ g / m l (Fig. 3). At this concentration of cytochalasin B, phagocytosis of OK-432 particles also was completely inhibited as revealed by microscopic observations (data not shown). These results strongly indicate that the OK-432-induced stimulation of arachidonic acid metabolism in macrophages is induced by phagocytosis of foreign bodies, OK-432 particles. When OK-432 was heat-treated (120°C for 20 min), stimulative effects on arachidonic acid metabolism was further increased than by non-treated OK-432 (Fig. 4). This increased activity was also inhibited completely by cytochalasin B treatment (Fig. 5), suggesting that the increase in arachidonic acid metabolism by heat-treated OK-432 is aslo induced by phagocytosis of OK-432 particles. Microscopic observations revealed that nontreated OK-432 particles easily form aggregates in the incubation medium, however, after heat treatment OK432 particles tend to disaggregate in the medium. Consequently, increased activity by heat-treated OK-432 to stimulate arachidonic acid metabolism (Fig. 4) might be due to the qualitative changes of OK-432, viz. the particles of heat-treated OK-432 remained to be more phagocytozable forms in the medium than the nontreated OK-432, which easily form aggregates that would be less phagocytozable than free OK-432 particles. Kawaguchi et al. [2] reported that mouse peritoneal macrophages collected after intraperitoneal injection of OK-432 showed increased motility, adhesiveness, interleukin-1 production, and inhibition of tumor cell growth in vitro. However, when heat-treated OK-432 (120°C for 20 min) was injected, the peritoneal macrophages showed no activity to induce inhibition of tumor cell growth and production of interleukin-1, but showed the increased motility and adhesiveness [2]. Consequently, stimulation of arachidonic acid metabolism by OK-432 might be related with increased motility and adhesiveness of macrophages but not with induction of inhibition of tumor cells growth and interleukin-1 production. However, the activation of macrophages as reflected by stimulation of arachidonic acid metabolism by OK-432 might play some significant roles in immunopotentiation that leads to antitumor effects demonstrated experimentally [4,13,20] and clinically [10,21-23]. Although the present work revealed that OK-432 has an activity to stimulate arachidonic
161 acid metabolism in macrophages, further studies are necessary to clarify mechanism of antitumor action of OK-432 in vivo.
Acknowledgement This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan. References l Okamoto, H., Shoin, S., Koshimura, S. and Shimizu, R. (1967) Jpn. J. Microbiol. 11,323-336. 2 Kawaguchi, T., Suematsu, M., Masuda-Koizumi, H., Mitsui, H., Suzuki, S., Matsuno, T., Ogawa, H. and Nomoto, K. (1983) Immunopharmacology 6, 177-189. 3 Uchida, A. and Micksche, M. (1983) Int. J. Cancer 31, 1-5. 4 Hojo, H. and Hashimoto, Y. (1981) Gann 72, 692-699. 5 Saito, M., Ebina, T., Koi, M., Yamaguchi, T., Kawade, Y. and Ishida, N. (1982) Cell. Immunol. 68, 187-192. 6 lchimura, O., Suzuki, S., Saito, M., Sugawara, Y. and Ishida, N. (1985) Int. J. lmmunopharmacol. 7, 263-270. 7 Yamamoto, A., Nagamuta, M., Usami, Y., Sugawara, N., Watanabe, Y., Niitsu, Y. and Urushizaki, I. (1986) Immunopharmacology 11, 79-86. 8 Sakurai, Y., Tsukagoshi, S., Satoh, H., Akiba, T., Suzuki, S. and Takagi, Y. (1972) Cancer Chemother. Rep. 56, 9-17.
9 Yamagishi, H., Pellis, N.R. and Kahan, B.D. (1980) Cancer Immunol. Immunother. 9, 63-67. 10 Uchida, A., and Hoshino, T. (1980) Cancer 45, 476-483. 11 Torisu, M., Katano, M., Kimura, H., Itoh, H. and Takesue, M. (1983) Surgery 93, 357-364. 12 Watanabe, Y. and lwa, T. (1984) Cancer 53, 248-253. 13 Ishii, Y., Yamamoto, H., Toh, K. and Kikuchi, K. (1976) Gann 67, 115-119. 14 Torikai, T., ]toh, O., Satoh, M. and Osawa, T. (1981)) Gann 71, 52-59. 15 Watanabe, M., Tamura, T., Ohashi, M., Hirasawa, N., Ozeki, T., Tsurufuji, S., Fujiki, H. and Ohuchi, K. (1990) Biochim. Biophys. Acta 1/)47, 141-147. 16 Ohuchi, K., Sugawara, T., Watanabe, M., Hirasawa, N., Tsurufuji, S., Fujiki, H., Sugimura, T. and Christensen, S.B. (1987) J. Cancer Res. Clin. Oncol. 113, 319-324. 17 Ohuchi, K., Watanabe, M., Hirasawa, N., Tsurufuji, S., Ozeki, T. and Fujiki, H. (1988) Biochim. Biophys. Acta 971, 85-91. 18 Ohuchi, K., Takahashi, C., Watanabe, M., Hirasawa, N., Suzuki, Y., Kudo, M., Konishi, T. and Tsurufuji, S. (1988) J. Clin. Lab. Immunol. 27, 171 178. 19 Zigmond, S.H. and Hirsch, J.G. (1972) Exp. Cell Res. 73,383-393. 20 Aoki, T., Kvedar, J.P., Hollis, V.W. Jr. and Bushar, G.S. (1976) J. Natl. Cancer Inst. 56, 687-690. 21 Kurokawa, T., Hanori, T. and Furue, H. (1972) Cancer Chemother. Rep. 56, 211-220. 22 Kimura, I., Ohnoshi, T., Yasuhara, S., Sugiyama, M., Urabe, Y., Fujii, M. and Machida, K. (1976) Cancer 37, 2201-221)3. 23 Tanaka, R., Sekiguchi, K., Suzuki, Y., Sobue, H. and Ueki, K. (1980) Cancer 46, 1688-1694.
