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Cancer Immunol Immunother (1990) 30:374-376
ancer mmunology mmunotherapy
© Springer-Verlag 1990
Short communication
Effects of opioid peptides on the cellular immunity in spleen cells from intact nude mice or nude mice bearing human ovarian carcinoma Yoshihiro Kikuchi, Tsunekazu Kita, and lchiro Nagata Department of Obstetrics and Gyne¢ology, National Defense Medical College, Namiki 3-2, Tokorozawa, Saitama 359, Japan
Summary. The present study was designed to explore the effects of opioid peptides on the lytic activity of spleen cells from intact nude mice or nude mice bearing human ovarian cancer cells (KF). When the spleen cells from intact nude mice were incubated with various concentrations of opioid peptides, the ability of the spleen cells to lyse the KF cells was significantly stimulated between 0.05 nM and 50 nM concentrations of all opioid peptides used in this study. The degree of stimulation was most marked at 5 nM opioid peptides and the most marked stimulatory effect was obtained by c~-endorphin. On the other hand, the lytic activity of spleen cells from nude mice challenged with the KF cells was about two-fold higher than that of intact nude mice, suggesting that spleen cells from nude mice challenged with KF cells have KF-cell-specific cytotoxicity. Even if the spleen cells were incubated with any concentration of c~-endorphin or [Met]enkephalin indicated, the lytic activity remained unchanged. In conträst, only B-endorphin resulted in a significant increase of the lytic activity between 0.5 nM and 50 nM. These results suggest that opioid peptides play a crucial role in immune surveillance mechanisms.
Introduction In the last few years, a new stream of medical research sterns from studies of the potential influence of opioid peptides on the immune system. In 1979, Wybran et al. [10] demonstrated the presence of [Met]enkephalin-like and morphin-like receptors on human blood lymphocytes, whereas Hazum et al. [3] demonstrated the presence of [~-endorphin receptors on human lymphoblastoid cell lines. In addition, it has been reported that ~-endorphin enhances the lymphocyte proliferative response to mitogens [2] and human peripheral blood lymphocyte natural killer activity [4]. One possible hypothesis stemming from these studies is that enkephalin and endorphins, which are produced in the central nervous system, may influence a variety of immune mechanisms, subsequently regulating tumor growth. Whether opiates and opioi6 peptides act directly or indirectly upon immune function remains to be determined.
Offprint requests to: Y. Kikuchi
We have established a human ovarian cancer cell line transplantable to nude mice and designated it "KF" [6]. We have also demonstrated that the KF cell proliferation was dose-dependently inhibited by endorphins and to less extent [Met]enkephalin [7]. Thus, in the present study we attempted to determine effects of opioid peptides on natutal or KF-cell-specific cytotoxicity in spleen cells of intact or tumor-bearing nude mice.
Materials and methods Agents. c~-Endorphin, I~-endorphin and [Met]enkephalin were obtained from Sigma Chemical Co., St. Louis, Miss. Animals. Female BALB/c nude mice, 6 weeks old, were obtained from Japan Clea Laboratories, Tokyo, Japan, and maintained in a pathogen-free environment. Cells. The human ovarian cancer cell line [6], designated KF, established from serous eystadenocarcinoma of the ovary, was exclusively used. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin (100 units/ml), and streptomycin (100 txg/ml) (Grand Island Biological Co.) in a 5% CO2 atmosphere at 37 ° C. The medium was changed every 3 days, and the cells were passed when confluency was achieved. Cytotoxicity assay. Spleen cells from intact nude mice were used as effector cells for a natural cytotoxicity assay. Spleen cells from nude mice 3 weeks after KF cell inoculation were used as effector cells to obtain KF-cell-specific cytotoxicity. The viable 5 x 10» KF cells were inoculated s.c. to the right flank of nude mice and 3 weeks after inoculation spleens from the nude mice were resected and used as effector cells. The spleen cells were prepared as described previously [5]. The KF cells in a pre-confluent state were used as target cells and were labeled with 100 gCi sodium [51Cr]chromate solution (New England Nuclear, Boston, Mass) for 1 h in 1 ml medium. After three washings, 104 cells in 0.1 ml medium were pipetted into U-bottomed microtiter plates (Nunc, Roskilde, Denmark). Various concentrations of effector cells in 0.1 ml medium were added in quadruplicate to give effector:target cells ratlos of 100: 1, 50:1 and 25:1, respectively. After incubation for 4 h at 37 ° C in a humidified atmosphere of 5% CO2 in air, supernatants were collected using a Titertek collection sys-
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Concentrations of opioid peptides(M)
Concentrations of opioid peptides (M)
Fig. 1. Effects of opioid peptides on natural cytotoxic]ty to K F cells in spleen cell from intact nude mice. The spleen cells preincubated with medium alone or 0.5 nM-0.05 l.tM concentrations of each opioid peptides for 24 h were used as control or test effector cells, respectively. Bars show the mean _+ SD from quadruplicate experiments. *P
Cancer Immunol Immunother (1990) 30:374-376
ancer mmunology mmunotherapy
© Springer-Verlag 1990
Short communication
Effects of opioid peptides on the cellular immunity in spleen cells from intact nude mice or nude mice bearing human ovarian carcinoma Yoshihiro Kikuchi, Tsunekazu Kita, and lchiro Nagata Department of Obstetrics and Gyne¢ology, National Defense Medical College, Namiki 3-2, Tokorozawa, Saitama 359, Japan
Summary. The present study was designed to explore the effects of opioid peptides on the lytic activity of spleen cells from intact nude mice or nude mice bearing human ovarian cancer cells (KF). When the spleen cells from intact nude mice were incubated with various concentrations of opioid peptides, the ability of the spleen cells to lyse the KF cells was significantly stimulated between 0.05 nM and 50 nM concentrations of all opioid peptides used in this study. The degree of stimulation was most marked at 5 nM opioid peptides and the most marked stimulatory effect was obtained by c~-endorphin. On the other hand, the lytic activity of spleen cells from nude mice challenged with the KF cells was about two-fold higher than that of intact nude mice, suggesting that spleen cells from nude mice challenged with KF cells have KF-cell-specific cytotoxicity. Even if the spleen cells were incubated with any concentration of c~-endorphin or [Met]enkephalin indicated, the lytic activity remained unchanged. In conträst, only B-endorphin resulted in a significant increase of the lytic activity between 0.5 nM and 50 nM. These results suggest that opioid peptides play a crucial role in immune surveillance mechanisms.
Introduction In the last few years, a new stream of medical research sterns from studies of the potential influence of opioid peptides on the immune system. In 1979, Wybran et al. [10] demonstrated the presence of [Met]enkephalin-like and morphin-like receptors on human blood lymphocytes, whereas Hazum et al. [3] demonstrated the presence of [~-endorphin receptors on human lymphoblastoid cell lines. In addition, it has been reported that ~-endorphin enhances the lymphocyte proliferative response to mitogens [2] and human peripheral blood lymphocyte natural killer activity [4]. One possible hypothesis stemming from these studies is that enkephalin and endorphins, which are produced in the central nervous system, may influence a variety of immune mechanisms, subsequently regulating tumor growth. Whether opiates and opioi6 peptides act directly or indirectly upon immune function remains to be determined.
Offprint requests to: Y. Kikuchi
We have established a human ovarian cancer cell line transplantable to nude mice and designated it "KF" [6]. We have also demonstrated that the KF cell proliferation was dose-dependently inhibited by endorphins and to less extent [Met]enkephalin [7]. Thus, in the present study we attempted to determine effects of opioid peptides on natutal or KF-cell-specific cytotoxicity in spleen cells of intact or tumor-bearing nude mice.
Materials and methods Agents. c~-Endorphin, I~-endorphin and [Met]enkephalin were obtained from Sigma Chemical Co., St. Louis, Miss. Animals. Female BALB/c nude mice, 6 weeks old, were obtained from Japan Clea Laboratories, Tokyo, Japan, and maintained in a pathogen-free environment. Cells. The human ovarian cancer cell line [6], designated KF, established from serous eystadenocarcinoma of the ovary, was exclusively used. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin (100 units/ml), and streptomycin (100 txg/ml) (Grand Island Biological Co.) in a 5% CO2 atmosphere at 37 ° C. The medium was changed every 3 days, and the cells were passed when confluency was achieved. Cytotoxicity assay. Spleen cells from intact nude mice were used as effector cells for a natural cytotoxicity assay. Spleen cells from nude mice 3 weeks after KF cell inoculation were used as effector cells to obtain KF-cell-specific cytotoxicity. The viable 5 x 10» KF cells were inoculated s.c. to the right flank of nude mice and 3 weeks after inoculation spleens from the nude mice were resected and used as effector cells. The spleen cells were prepared as described previously [5]. The KF cells in a pre-confluent state were used as target cells and were labeled with 100 gCi sodium [51Cr]chromate solution (New England Nuclear, Boston, Mass) for 1 h in 1 ml medium. After three washings, 104 cells in 0.1 ml medium were pipetted into U-bottomed microtiter plates (Nunc, Roskilde, Denmark). Various concentrations of effector cells in 0.1 ml medium were added in quadruplicate to give effector:target cells ratlos of 100: 1, 50:1 and 25:1, respectively. After incubation for 4 h at 37 ° C in a humidified atmosphere of 5% CO2 in air, supernatants were collected using a Titertek collection sys-
375
,
40 o
~
.-~ 40
30
:: 30
.fi 20
20
D.
10
a.
0
*
10
5XI0-1°SXI0 -9 5X10-8 5X10-7
5Xl0-a05Xl0-9 5X10-8 5XI0 -7
Concentrations of opioid peptides(M)
Concentrations of opioid peptides (M)
Fig. 1. Effects of opioid peptides on natural cytotoxic]ty to K F cells in spleen cell from intact nude mice. The spleen cells preincubated with medium alone or 0.5 nM-0.05 l.tM concentrations of each opioid peptides for 24 h were used as control or test effector cells, respectively. Bars show the mean _+ SD from quadruplicate experiments. *P
