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Generation of leukotrienes and hydroxyeicosatetraenoic acids in peritoneal macrophages of tumor-bearing mice
Prcstagiandins Leukotrienes
and Essential Fatty Acids (1991) 44, 185-190 @ Longman Group UK Ltd 1991
Generation of Leukotrienes and Hydroxyeicosatetraenoic Peritoneal Macrophages of Tumor-bearing Mice T. Hidaka, M. Tsuruta*,
Acids in
Y. Tom&a*, T. Inokuchi*, M. Sugiyama and R. Ogura
Departments of Medical Biochemistry and *Public Health, and ‘Institute of Medical Mass Spectrometry, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830, Japan (Reprint requests to TH)
To examine the potential role of lipoxygenase products in the pathophysioiogy observed after experimental tumor implantation, we examined the generation of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) in peritoneal macrophages. C57BIj6 mice were given subcutaneous inoculations of B16 melanoma ceils, and peritoneal macrophages were isolated various days after the inoculation. Macrophages were incubated for 1 h at 37°C in serum-free RPMI1640 containing 10 PM calcium ionophore A23187, 10 PM exogenous arachidonic acid (AA), 5 mM cystehre hydrochloride and 1 mM reduced giutathione. LTs and HETEs were separately extracted, passed through Sep-Pak cartridges, then identified and quantitated with a HPLC system using W absorbance. The B16 mehmoma-ceil-treated/ untreated macrophages were found to produce substantial amounts of IS-HETE, 124IETE and 5-HETE and LTC4 by enzymatic mechanisms. Thus, when determined under various conditions, the production of HETEs was dependent on substrate-concentration, incubation-time and cell-number. The production of LTC, was dependent on incubation-time and ceii number but not substrate-concentration, indicating utihition of endogenous AA stores. Of these products, 12-HETE and LTC4 showed a significant increase on the fourth day after the tumor ceil hrocuiation and returned to the control level by the 11th day after the same treatment. These results suggest that in vivo tumor cell implantation may induce a transient increase of 12.HETE and LTC, production in macrophages.
ABSTRACT.
INTRODUCTION
the effect of tumor cell implantation on the capacities of mouse peritoneal macrophages to generate HETEs and LTs, using C57BL/6 mice and syngeneic B16 melanoma cells.
Macrophages produce large amounts of prostaglandins (PGs) and leukotrienes (LTs). Of the various prostanoids, PGE2, is involved in macrophage differentiation (1, 2) and also plays an immunosuppressive role by stimulating suppressor T-cells as a cytokine (3-5). However, immunological roles of LTs remain unclear. The capacity of macrophages to generate LTs develops at a relatively late stage of their differentiation (6). It is conceivable that the generated LTs are involved in functions of mature macrophages rather than in their differentiation process. Macrophages are known to recognize and suppress or kill tumor cells directly and indirectly (7, 8). However the relationship between this process and LTs or hydroxylated arachidonic acids (HETEs) remains obscure. Thus, we examined
MATERIALS AND METHODS Materials RPM11640 powder medium was purchased from Nissui Pharmaceutical Co Ltd, Tokyo. Sterile penicillin-streptomycin solution and fetal bovine serum (FBS) were purchased from Gibco Laboratories, New York. Calcium ionophore A23187 was obtained from Calbiochem Corp, LaJolla, CA. Arachidonic acid (AA) (purity % 99.5%) was purchased from NuCheck Prep, Elysian, MN. Synthetic LTB4 and 5HETE were gifts from Ono Pharmaceutical Co, Osaka, Japan. Mono-HETE mixture including S(S,R)-HETE, 12(S)-HETE and 15(S)-HETE and cysteinyl LT mixture including LTC,, LTD4 and LTE, were purchased from Biomol. Res. Lab. Ins.,
Date received 18 May 1991 Date accepted 3 July 1991 185
186 Prostaglandins Leukotrienes and Essential Fatty Acids
PA. 13(S)-HODE ((9Z,llE,13(S))-13-hydroxy octadecadienoic acid) was purchased from Cayman Chemical Co, Michigan. Reduced glutathione (GSH), cysteine hydrochloride and EDTA(2Na) were purchased from Wako Pure Chem. Indust. Co Ltd. SepPak Cl8 cartridges were purchased from Waters Associates. All the solvents used were of highpressure liquid chromatography (HPLC) grade. Care and tumor-cell treatment of animals C57BL/6CR female mice, 7-8 weeks old, were housed in cages with wire mesh bottoms and fed standard lab chow and tap water ad libitum throughout the experimental period. All animals were maintained in an animal care facility at a room temperature of 20-22°C. B16 melanoma cells (2 X lo6 cells) suspended in saline (1 x 10’ cells/ml) were subcutaneously inoculated into the chest of the mouse. Control groups were prepared by injecting saline only. Preparation of macrophages At various days after tumor inoculation, peritoneal cells were harvested by washing the peritoneal cavity with cold phosphate-buffered saline (PBS) immediately after killing the animals by cervical dislocation. Three days before cell harvest, 2 ml of sterile 10% proteose peptone in PBS was injected intraperitoneally. Havested cells were centrifuged at 100 x g for 10 min, resuspended in RPM1 1640 medium supplemented with 2 mM L-glutamine, 1% penicillin-streptomcycin and 10% heat-inactivated fetal bovine serum (FBS), and plated onto plastic tissue culture dishes (2 x lo6 cells/ml, 4 ml/dish). After 2-3 h at 37°C in 5% CO*, cultures were washed three times with Ca2+- and Mg2+-free PBS to remove nonadherent cells. The adherent cells were scraped out with a rubber policeman, resuspended in RPM11940 containing 10% FBS and added to 60 mm 4 Falcon plastic culture dishes at 3 x lo6 cells/4 ml/dish. More than 98% of the adherent cells were found to be viable by trypan blue exclusion test. Cells were cultured overnight at 37°C in a humidified chamber containing 5% C02. Incubation, purification and analysis of HETEs and LTs After being washed twice with 5 ml fresh serum-free RPM1 1640, cells were equilibrated for 60 min in 3 ml serum-free medium. Macrophages were incubated for 1 h at 37°C in 4 ml of serum-free RPM11640 containing exogeneous AA (10 PM), 10 FM calcium ionophore A23187, 5 mM cysteine hydrochloride and 1 mM reduced glutathione, unless otherwise stated. The reaction mixture was constructed by a slight modification of Lokesh’s
method (9). Reactions were terminated by transferring the medium to a tube containing 10 ml cold methanol, and by adding 2 ml cold methanol to the adherent cells. Cells were removed from the plate utilizing a rubber policeman and mixed with the corresponding medium sample. The mixed sample was sonicated for 30 s at 4°C and centrifuged for 15 min at 3000 rpm. The pellet was subjected to analyses of esterified HETEs. 13-HODE (50 ng) was added to the supernatant as an internal standard for the HETEs analyses. Obtained samples were dried under vacuum using a Speed-Bat lyophilizer. HETEs and LTs were extracted by the method of Miyamoto et al (10). Briefly, the dried residue was mixed with 2.5 ml saline and extracted with 4 volumes of Folch’s solution (chloroform/ methanol = 2/l, v/v). PGB2 (60 ng) was added to the upper phase as an internal standard. The two phases were separately evaporated and dissolved in 200 ~1 methanol. The upper phase residue in 200 ~1 methanol (cysteinyl LT fraction) was mixed with 5 ml distilled water, and applied to a Sep-Pak Cl, cartrigde column, which was successively eluted with 5 ml water, 10 ml 20% methanol in water and 10 ml 80% methanol. The 80% methanol fraction was collected for further analysis. The lower phase (LTB4 and HETE fraction) dissolved in 200 ,c~l methanol was vigorously mixed with 15 ml water, and applied to a Sep-Pak Cis cartridge column, which was successively eluted with 5 ml water, 10 ml 20% methanol in water, 10 ml n-hexane and 10 ml acetone. The acetone fraction was collected for further analysis. The collected samples (80% methanol and acetone fractions) were dried under a Nz gas stream, and analyzed with a computed HPLC system (Hitachi) consisting of an L-6200 intelligent pump, an L-3000 photo diode array detector and a D-6000 Data station HPLC manager. Separation was achieved on a TSK-gel ODS-120T reverse phase column (Tosoh Co, 4.6 X 250 mm). The mobile phase for HETEs was a mixture of methanol/water/acetic acid (75 : 25 :O.Ol, v/v/v) (A) and for LTs, a mixture of methanol/ water/acetic acid (65:35:0.08, v/v/v) containing 0.5% EDTA (2Na) and 10% NH,OH up to pH 5.2 (B). The flowrate was 1 ml/min. LTs and HETEs were identified by retention time compared to authentic standards and by their ultraviolet absorption spectra obtained in the diode array spectrophotometric detector, and were quantitated by their absorbances using absorption coefficients of authentic standards.
RESULTS The following characterization was done using peritoneal machrophages obtained from tumor-celluntreated mice. Figure 1 shows typical chromatograms of hydroxy fatty acids (Fig. 1A) and cysteinyl
HETEs and LTs in Peritoneal Macrophages of Tumor-bearing
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Fig. 1 Typical chromatograms of hydroxy fatty acids (A) and cysteinyl LTs (B) produced from mouse peritoneal macrophages. Proteose peptone-elicited peritoneal macrophages from C57BL/6CR mice were incubated for 1 h at 37°C in 4 ml of serum-iree RPM11640 containing exogeneous AA (10 PM), 10 &I calcium ionophore A23187, 5 mM cysteine hydrochloride and 1 mM reduced glutathione. Reactions were terminated with methanol. The reaction mixture was dried after addition of IS-HODE as an internal standard. The dried residue was mixed with 2.5 ml saline and extracted with 4 volumes of chloroform/methanol (2/l, v/v). PGB, (60 ng) was added to the upper phase as another internal standard. HETEs and cysteinyl LTs were extracted from the lower and upper phases, respectively, using Sep-Pak C,, cartridge columns. Separation was achieved on a TSK-gel ODS- 120T reverse phase column (Tosoh Co, 4.6 x 250 mm). The mobile phase for HETEs was a mixture of methanol/water/acetic acid (75:25:0.01, v/v/v) (A) and for LTs, a mixture of methanoi/ water/acetic acid (65:35:0.08, v/v/v) containing 0.5% EDTA (2Na) and 10% NH,OH up to pH 5.2 (B). The flow-rate was 1 ml/min. Detection was done by a diode array spectrophotometer. The insets show typical UV absorption spectra of the materials (I, II, III and IV). Abbreviations: 15-, 12- and 5-HETEs = 15-, 12- and 5-hydroxyeicosatetraenoic acids; PGB, = prostaglandin BZ; I.S. = internal standard.
Mice
I87
LTs (Fig. 1B) produced from the macrophages by A23187 stimulation. HPLC analysis of the HETE and LTB4 fraction at 235 nm showed 3 major peaks (I, II and III) in addition to the internal standard 13-HODE(IS). There was no detectable peak corresponding to LTB4 as monitored at 270 nm. Analysis of the cysteinyl LT fraction at 280 nm showed only one peak (IV) at the retention volume corresponding to LTC4 without any peaks for LTD4 and LTE4. The insets in Figure 1 show the UV absorption spectra of these four peaks (I, II, III and IV), as analysed with the diode array spectrophotometric detector, which were completely consistent with those of 15HETE, ZZHETE, 5-HETE and LTC4, respectively. The time courses of 12-HETE, 15HETE, 5HETE and LTC4 generation by the macrophages after A23187 stimulation are shown in Figures 2A and 2B. 1ZHETE and LTC, increased time-dependently up to 60 min, while 15- and 5-HETEs increased time-dependently for up to 30 min, but reached a plateau thereafter. The generation of the HETEs declined after 60 min. The generation of these products was dependent on cell number (Figs 2C, 2D). The dependency of each product on exogenous arachidonate is shown in Figures 2E and 2F. All the HETEs increased with increasing concentrations of the exogenous substrate. However, the generation of LTC4 was not dependent on the concentration of exogenous arachidonate. Figure 3 shows the effects of B16 melanoma cellbearing conditions on HETE and LT generation capacities. 1ZHETE and LTC4 each showed a significant increase at the fourth day after the tumor cell inoculation and returned to the control level by the 11th day after the same treatment. 15-HETE showed the same tendency, while 5-HETE showed an increasing tendency at the 11th day after the tumor-cell inoculation. However, the latter two tendencies did not reach statistically significant levels.
DISCUSSION HETEs are easily produced by nonenzymatic processes unlike cysteinyl LTs which are generated only by the aid of a series of enzymes. HETE generation in this study was dependent on exogenous substrate concentration, incubation time and cell number, thereby indicating that our method appropriately evaluates enzyme-mediated generation of HETEs. LTC4 generation after A23187 stimulation was also dependent on incubation time and cell number, but not on exogenous substrate concentration. Thus, LTC4 generation was mostly due to endogenous supply of the substrate from cellular membranes. Our present study indicates that lZHETEand LTC4-generating capacities are significantly in-
188 Prostaglandins Leukotrienes
and Essential Fatty Acids
“9 t 1.5x10* cells
INCUBATION TIME (min)
CELL NUMBER ngfh ng/h/4xlO'
cells
TjL!L__ 1.5
3.0
4.5
CELL NUMBER (x1o’ cel’s)
Fig. 2 Time, cell-number and exogenous-substrate dependencies of HETE and LTC, generations from macrophages. A,B: time courses, CD: cell number dependency and E,F: exogenous substrate dependency; A,C,E: for HETEs and B,D,F: for LTC,. The generation was examined using the same reaction method as in the legend of Figure 1, except for various reaction times in A B, various cell numbers in C,D or various arachidonate concentrations in E,F. HETE reduction is expressed as r&.5 x lo1 cells in A, ngih in C and ng/2 X IOr’cells /h in E, and LTC, production as ng/4 x 10? cells m B, n&t in D and n&/4 X lo6 cells in F.
DAYS
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Fig. 3 Effects of B16 melanoma cell-bearing conditions on HETE and LT generation capacities. B16 melanoma cells (2 X 10” cells) in saline were subcutaneously inoculated into the chest. Saline only was injected for O-day controls. At various days after tumor inoculation, peritoneal macrophages were obtained and analyzed in the same way as described in the legend of Figure 1. HETEs and LTs were quantitated by their absorbances using absorption coefficients of authentic standards. 12-HETE ressed as ngh/2 X 10 ceils; 15HETE and 5-HETE as X 10” cells and LTC, as t&r/8 x 10” ceils. Data are :he means + S.D. of five separate experiments. *: p < 0.05.
creased on the fourth day after the tumor inoculation, and return to the control level by the 11th day. Interestingly, such an increase in the early stage after tumor inoculation can also be seen in the generation capacity of cyclooxygenase products like PGE2 and 6-keto-PGF1, (11, 12). However, later time-courses appear to be different in that the enhanced capacity for the synthesis was transient for 1ZHETE and LTC4, but was persistent for the cyclooxygenase products (12). The enhanced PG production in tumor-bearing macrophages was explained by enhanced conversion efficiency of arachidonic acid into PGs (11). The mechanism or enzymes involved in the temporary increase in the generation capacity of 1ZHETE and LTC4 in the early stage were not elucidated in this study. 12-HETE has been reported to induce chemotaxis for neutrophils, degranulation of neutrophils, chemokinesis for smooth muscle cells and an increase in mononuclear procoagulant activity (13). Enhanced production of IZHETE after tumor inoculation was also demonstrated with rat alveolar
HETEs and LTs in Peritoneal Macrophages of Tumor-bearing
macrophages by Kort et al and Vincent et al (14, 15). Among lipoxygease products, LTB4 has been mostly mentioned in relation to cytokine production and tumor-growth inhibition (16-21). However, in mouse peritoneal macrophages the production of LTB4 appears to be negligible, if present at all, as compared with LTC4 which is produced in large amounts. LTC4 has been reported to induce interferon-y output from T-cells (16, 19), along with or thereby stimulating the release of interleukin 1 or 2 (8, 16, 18, 19) and tumor necrosis factor (8). Such cytokine production was enhanced by a cyclooxygenase inhibitor (20) while it was attenuated by lipoxygenase inhibitors (19). LTC4 also enhances macrophage cytostatic activity by the aid of increased cytosolic Ca*+ (22) or after metabolic conversion (21). The inhibition of 5lipoxygenase of peritoneal macrophages activated by A23187 was demonstrated to have the effect of inhibiting the cytotoxic activity towards tumor cells (23). Oxygen radicals produced by lipoxygenation have been related to natural killer cell-mediated cytotoxicity (24). Likewise, in macrophages it would be possible that lipoxygenation processes leading to LTC4, 12HETE or other lipoxygenase products release cytotoxic oxygen radicals. Theoretically, macrophages, natural killer T-cells and granulocytes migrating to tumor cells could exhibit anti-tumor suppressive activities. Thus, it appears that the generation of LTC4 and probably 12-HETE may bring about suppressive functions on tumor progression, in contrast with PGE?, which augments both tumor-mediated immunosuppression and tumor growth. The increase in lipoxygenase products shown in our present study may induce a positive immume response. However, this positive response may be masked by the immunosuppressive action of PGE?, produced in large amounts after tumor implantation. Acknowledgements We thank Ono Pharmaceutical Company (Osaka, Japan) for providing a generous supply of synthetic LTs, HETEs and PGBz. This work was partly supported by grants from the Ministry of Education, Science and Culture of Japan (No. 63570462)
References I. Honma Y, Kasukabe T. Hozumi M, Koshihara Y. Regulation of prostaglandin synthesis during differentiation of cultured mouse myeloid leukemia cells. J. Cell Physiol., 104: 349-357. 1980. 2. Honma Y, Kasukabe T, Hozumi M. Inhibition of differentiation of cultured mouse myeloid leukemia cells by nonsteroidal antiinflammatory agents and counteraction of the inhibition by prostaglandin E,. Cancer Res., 39: 2190-2194, 1979. 3. Goodwin J S, Bankhurst A D, Messner R P. Suppression of human T-cell mitogenesis by prostaglandin. Existence of a prostaglandin-producing suppressor cell. J. Exp. Med. 146: 1719-1734. 1977.
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4. Maca R D. The effects of prostaglandins on the proliferation of cultured human T lymphocytes. Immunopharmacol. 6: 267-277,1983. 5. Chouaib’S, Chatenoud L, Klatzmann D. Fradelizi D. The mechanisms of inhibition of human IL 2 production. PG2, induction of suppressor T lymphocytes. J. immunol., 132, 1’831-1857, 1984. 6. Koehler L. Hass R. Wessel K. Dewitt D L. Kaever V.‘Resch i, Goppelt-&uebe M. Atiered arachidonic acid metabolism during differentiation of the human monoblastoid cell line U937. Biochim Bioohvs Acta. 1042: 395-403. 1990. 7. Fidier’I J, Kleinerman E S. iymphokine-activated human blood monocytes destroy tumor cells but not normal cells under cocultivation conditions. J. Clinical Oncology, 2: 937-943, 1984. 8. Philip R. Epstein L B. Tumour necrosis factor as immunomodulator and mediator of monocyte cytotoxicity induced by itself, y-interferon and interleukin-1. Nature 323: 86-89, 1986. 9. Lokesh B R, German B, Kinsella .I E. Differential effects of docosahexaenoic acid and eicosapentaenoic acid on suppression of lipoxygenase pathway in peritoneal macrophages. Biochim. Biophys. Acta 958: 99-107, 1988. IO. Miyamoto T. Lindgren J A. Samuelsson, B. Isolation and identification of lipoxygenase products from the rat central nervous system. Biochim. Biophys. Acta 922: 372-378, 1987. II. Pelus L M, Bockman R S. Increased prostaglandin synthesis by macrophages from tumor-bearing mice. J. Immunol., 123, 2118-2125, 1979. 12. Plescia 0 J, Pontieri G M, Brown J, Racis S. Ippoliti F, Bellelli L, Sezzi M L, Lipari M. Amplification by macrophages of prostaglandin-mediated immunosuppression in mice bearing syngeneic tumors. Prostaglandins, Leukotrienes and Medicine, 16, 205-223, 1984. 13 Stenson W F, Parker C W. Prostaglandins, macrophages. and immunity. J. Immunol., 125, l-5, 1980. 14 Kort W J. Zijlstra F J, Weijma I M. Eicosanoid synthesis of alveolar macrophages in rats with malignant mammary tumors: Differences in rats treated with and without carrageenan implants. Prostaglandins Leukotrienes and Essential Fatty Acids, 37: 113-120, 1989. 15 Vincent J E, Vermeer M A, Kort W J, Zijlstra F J. The formation of thromboxane B,, leukotriene B, and 12-hydroxyeicosatetraenoic aid by alveolar macrophages after activation during tumor growth in the rat. Biochim. Biophys. Acta, 1042, 255-2.58. 1990. 16 Johnson H M, Torres B A. Leukotrienes, positive signals for regulation of interferon-y production. J Immunol 132: 413-416, 1984. 17 Rola-Pleszczynski M. Differential effects of Leukotriene B, on T4+ and T8’ lymphocyte phenotype and immunoregulatory functions. 1 Immunol 135: 1357-1360, 1985. 18. Rola-Pleszczynski M. Lemarire 1. Leukotrienes augment interleukin production by human monocvtes. J Immunol 135: 3958-3961. 1985. 19. Farrar -W L, Humes J L. The role of arachidonic acid metabolism in the activities of interleukin 1 and 2. J Immunol 135: 1153-1159, 1986. 20. Rola-Pleszczynski M, Bouvrette L, Gingras D, Girard M. Identification of interferon-y as the lymphokine that mediates leukotriene Bcinduced immunoregulation. J Immunol 139: 513-517. 1987. 21. Gagnon L. Filion L G, Dubois C, Rola-Pleszczynski, M. Leukotrienes and macrophage activation: Augmented cytotoxic activity and enhanced interleukin 1, tumor necrosis factor-and hydrogen peroxide production. Agents and Actions, 26, 141-147, 1989. 22. Van Hilten J A, Ben Efraim S, Zijlstra F J, Bonta 1 L. Leukotriene C, is an essential 5-lipoxygenase
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intermediate in A23187-induced macrophage cytostatic activity against P815 tumor cells. Prostaglandins Leukotrienes and Essential Fatty Acids, 39: 283-290, 1990. 23. Van Hilten J A, Elliott G R, Bonta I L. Specific lipoxygenase inhibition reverses macrophage cytostasis towards P815 tumor cells in vitro induced
by the calcium ionophore A23187. Prostaglandins Leukotrienes and Essential Fatty Acids, 34: 187-192,1988. 24. Bray R A, Brahmi Z. Role of lipoxygenaton in human natural killer cell activation. J. Immunol. 136: 1783-1790,1986.
and Essential Fatty Acids (1991) 44, 185-190 @ Longman Group UK Ltd 1991
Generation of Leukotrienes and Hydroxyeicosatetraenoic Peritoneal Macrophages of Tumor-bearing Mice T. Hidaka, M. Tsuruta*,
Acids in
Y. Tom&a*, T. Inokuchi*, M. Sugiyama and R. Ogura
Departments of Medical Biochemistry and *Public Health, and ‘Institute of Medical Mass Spectrometry, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830, Japan (Reprint requests to TH)
To examine the potential role of lipoxygenase products in the pathophysioiogy observed after experimental tumor implantation, we examined the generation of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) in peritoneal macrophages. C57BIj6 mice were given subcutaneous inoculations of B16 melanoma ceils, and peritoneal macrophages were isolated various days after the inoculation. Macrophages were incubated for 1 h at 37°C in serum-free RPMI1640 containing 10 PM calcium ionophore A23187, 10 PM exogenous arachidonic acid (AA), 5 mM cystehre hydrochloride and 1 mM reduced giutathione. LTs and HETEs were separately extracted, passed through Sep-Pak cartridges, then identified and quantitated with a HPLC system using W absorbance. The B16 mehmoma-ceil-treated/ untreated macrophages were found to produce substantial amounts of IS-HETE, 124IETE and 5-HETE and LTC4 by enzymatic mechanisms. Thus, when determined under various conditions, the production of HETEs was dependent on substrate-concentration, incubation-time and cell-number. The production of LTC, was dependent on incubation-time and ceii number but not substrate-concentration, indicating utihition of endogenous AA stores. Of these products, 12-HETE and LTC4 showed a significant increase on the fourth day after the tumor ceil hrocuiation and returned to the control level by the 11th day after the same treatment. These results suggest that in vivo tumor cell implantation may induce a transient increase of 12.HETE and LTC, production in macrophages.
ABSTRACT.
INTRODUCTION
the effect of tumor cell implantation on the capacities of mouse peritoneal macrophages to generate HETEs and LTs, using C57BL/6 mice and syngeneic B16 melanoma cells.
Macrophages produce large amounts of prostaglandins (PGs) and leukotrienes (LTs). Of the various prostanoids, PGE2, is involved in macrophage differentiation (1, 2) and also plays an immunosuppressive role by stimulating suppressor T-cells as a cytokine (3-5). However, immunological roles of LTs remain unclear. The capacity of macrophages to generate LTs develops at a relatively late stage of their differentiation (6). It is conceivable that the generated LTs are involved in functions of mature macrophages rather than in their differentiation process. Macrophages are known to recognize and suppress or kill tumor cells directly and indirectly (7, 8). However the relationship between this process and LTs or hydroxylated arachidonic acids (HETEs) remains obscure. Thus, we examined
MATERIALS AND METHODS Materials RPM11640 powder medium was purchased from Nissui Pharmaceutical Co Ltd, Tokyo. Sterile penicillin-streptomycin solution and fetal bovine serum (FBS) were purchased from Gibco Laboratories, New York. Calcium ionophore A23187 was obtained from Calbiochem Corp, LaJolla, CA. Arachidonic acid (AA) (purity % 99.5%) was purchased from NuCheck Prep, Elysian, MN. Synthetic LTB4 and 5HETE were gifts from Ono Pharmaceutical Co, Osaka, Japan. Mono-HETE mixture including S(S,R)-HETE, 12(S)-HETE and 15(S)-HETE and cysteinyl LT mixture including LTC,, LTD4 and LTE, were purchased from Biomol. Res. Lab. Ins.,
Date received 18 May 1991 Date accepted 3 July 1991 185
186 Prostaglandins Leukotrienes and Essential Fatty Acids
PA. 13(S)-HODE ((9Z,llE,13(S))-13-hydroxy octadecadienoic acid) was purchased from Cayman Chemical Co, Michigan. Reduced glutathione (GSH), cysteine hydrochloride and EDTA(2Na) were purchased from Wako Pure Chem. Indust. Co Ltd. SepPak Cl8 cartridges were purchased from Waters Associates. All the solvents used were of highpressure liquid chromatography (HPLC) grade. Care and tumor-cell treatment of animals C57BL/6CR female mice, 7-8 weeks old, were housed in cages with wire mesh bottoms and fed standard lab chow and tap water ad libitum throughout the experimental period. All animals were maintained in an animal care facility at a room temperature of 20-22°C. B16 melanoma cells (2 X lo6 cells) suspended in saline (1 x 10’ cells/ml) were subcutaneously inoculated into the chest of the mouse. Control groups were prepared by injecting saline only. Preparation of macrophages At various days after tumor inoculation, peritoneal cells were harvested by washing the peritoneal cavity with cold phosphate-buffered saline (PBS) immediately after killing the animals by cervical dislocation. Three days before cell harvest, 2 ml of sterile 10% proteose peptone in PBS was injected intraperitoneally. Havested cells were centrifuged at 100 x g for 10 min, resuspended in RPM1 1640 medium supplemented with 2 mM L-glutamine, 1% penicillin-streptomcycin and 10% heat-inactivated fetal bovine serum (FBS), and plated onto plastic tissue culture dishes (2 x lo6 cells/ml, 4 ml/dish). After 2-3 h at 37°C in 5% CO*, cultures were washed three times with Ca2+- and Mg2+-free PBS to remove nonadherent cells. The adherent cells were scraped out with a rubber policeman, resuspended in RPM11940 containing 10% FBS and added to 60 mm 4 Falcon plastic culture dishes at 3 x lo6 cells/4 ml/dish. More than 98% of the adherent cells were found to be viable by trypan blue exclusion test. Cells were cultured overnight at 37°C in a humidified chamber containing 5% C02. Incubation, purification and analysis of HETEs and LTs After being washed twice with 5 ml fresh serum-free RPM1 1640, cells were equilibrated for 60 min in 3 ml serum-free medium. Macrophages were incubated for 1 h at 37°C in 4 ml of serum-free RPM11640 containing exogeneous AA (10 PM), 10 FM calcium ionophore A23187, 5 mM cysteine hydrochloride and 1 mM reduced glutathione, unless otherwise stated. The reaction mixture was constructed by a slight modification of Lokesh’s
method (9). Reactions were terminated by transferring the medium to a tube containing 10 ml cold methanol, and by adding 2 ml cold methanol to the adherent cells. Cells were removed from the plate utilizing a rubber policeman and mixed with the corresponding medium sample. The mixed sample was sonicated for 30 s at 4°C and centrifuged for 15 min at 3000 rpm. The pellet was subjected to analyses of esterified HETEs. 13-HODE (50 ng) was added to the supernatant as an internal standard for the HETEs analyses. Obtained samples were dried under vacuum using a Speed-Bat lyophilizer. HETEs and LTs were extracted by the method of Miyamoto et al (10). Briefly, the dried residue was mixed with 2.5 ml saline and extracted with 4 volumes of Folch’s solution (chloroform/ methanol = 2/l, v/v). PGB2 (60 ng) was added to the upper phase as an internal standard. The two phases were separately evaporated and dissolved in 200 ~1 methanol. The upper phase residue in 200 ~1 methanol (cysteinyl LT fraction) was mixed with 5 ml distilled water, and applied to a Sep-Pak Cl, cartrigde column, which was successively eluted with 5 ml water, 10 ml 20% methanol in water and 10 ml 80% methanol. The 80% methanol fraction was collected for further analysis. The lower phase (LTB4 and HETE fraction) dissolved in 200 ,c~l methanol was vigorously mixed with 15 ml water, and applied to a Sep-Pak Cis cartridge column, which was successively eluted with 5 ml water, 10 ml 20% methanol in water, 10 ml n-hexane and 10 ml acetone. The acetone fraction was collected for further analysis. The collected samples (80% methanol and acetone fractions) were dried under a Nz gas stream, and analyzed with a computed HPLC system (Hitachi) consisting of an L-6200 intelligent pump, an L-3000 photo diode array detector and a D-6000 Data station HPLC manager. Separation was achieved on a TSK-gel ODS-120T reverse phase column (Tosoh Co, 4.6 X 250 mm). The mobile phase for HETEs was a mixture of methanol/water/acetic acid (75 : 25 :O.Ol, v/v/v) (A) and for LTs, a mixture of methanol/ water/acetic acid (65:35:0.08, v/v/v) containing 0.5% EDTA (2Na) and 10% NH,OH up to pH 5.2 (B). The flowrate was 1 ml/min. LTs and HETEs were identified by retention time compared to authentic standards and by their ultraviolet absorption spectra obtained in the diode array spectrophotometric detector, and were quantitated by their absorbances using absorption coefficients of authentic standards.
RESULTS The following characterization was done using peritoneal machrophages obtained from tumor-celluntreated mice. Figure 1 shows typical chromatograms of hydroxy fatty acids (Fig. 1A) and cysteinyl
HETEs and LTs in Peritoneal Macrophages of Tumor-bearing
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Fig. 1 Typical chromatograms of hydroxy fatty acids (A) and cysteinyl LTs (B) produced from mouse peritoneal macrophages. Proteose peptone-elicited peritoneal macrophages from C57BL/6CR mice were incubated for 1 h at 37°C in 4 ml of serum-iree RPM11640 containing exogeneous AA (10 PM), 10 &I calcium ionophore A23187, 5 mM cysteine hydrochloride and 1 mM reduced glutathione. Reactions were terminated with methanol. The reaction mixture was dried after addition of IS-HODE as an internal standard. The dried residue was mixed with 2.5 ml saline and extracted with 4 volumes of chloroform/methanol (2/l, v/v). PGB, (60 ng) was added to the upper phase as another internal standard. HETEs and cysteinyl LTs were extracted from the lower and upper phases, respectively, using Sep-Pak C,, cartridge columns. Separation was achieved on a TSK-gel ODS- 120T reverse phase column (Tosoh Co, 4.6 x 250 mm). The mobile phase for HETEs was a mixture of methanol/water/acetic acid (75:25:0.01, v/v/v) (A) and for LTs, a mixture of methanoi/ water/acetic acid (65:35:0.08, v/v/v) containing 0.5% EDTA (2Na) and 10% NH,OH up to pH 5.2 (B). The flow-rate was 1 ml/min. Detection was done by a diode array spectrophotometer. The insets show typical UV absorption spectra of the materials (I, II, III and IV). Abbreviations: 15-, 12- and 5-HETEs = 15-, 12- and 5-hydroxyeicosatetraenoic acids; PGB, = prostaglandin BZ; I.S. = internal standard.
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LTs (Fig. 1B) produced from the macrophages by A23187 stimulation. HPLC analysis of the HETE and LTB4 fraction at 235 nm showed 3 major peaks (I, II and III) in addition to the internal standard 13-HODE(IS). There was no detectable peak corresponding to LTB4 as monitored at 270 nm. Analysis of the cysteinyl LT fraction at 280 nm showed only one peak (IV) at the retention volume corresponding to LTC4 without any peaks for LTD4 and LTE4. The insets in Figure 1 show the UV absorption spectra of these four peaks (I, II, III and IV), as analysed with the diode array spectrophotometric detector, which were completely consistent with those of 15HETE, ZZHETE, 5-HETE and LTC4, respectively. The time courses of 12-HETE, 15HETE, 5HETE and LTC4 generation by the macrophages after A23187 stimulation are shown in Figures 2A and 2B. 1ZHETE and LTC, increased time-dependently up to 60 min, while 15- and 5-HETEs increased time-dependently for up to 30 min, but reached a plateau thereafter. The generation of the HETEs declined after 60 min. The generation of these products was dependent on cell number (Figs 2C, 2D). The dependency of each product on exogenous arachidonate is shown in Figures 2E and 2F. All the HETEs increased with increasing concentrations of the exogenous substrate. However, the generation of LTC4 was not dependent on the concentration of exogenous arachidonate. Figure 3 shows the effects of B16 melanoma cellbearing conditions on HETE and LT generation capacities. 1ZHETE and LTC4 each showed a significant increase at the fourth day after the tumor cell inoculation and returned to the control level by the 11th day after the same treatment. 15-HETE showed the same tendency, while 5-HETE showed an increasing tendency at the 11th day after the tumor-cell inoculation. However, the latter two tendencies did not reach statistically significant levels.
DISCUSSION HETEs are easily produced by nonenzymatic processes unlike cysteinyl LTs which are generated only by the aid of a series of enzymes. HETE generation in this study was dependent on exogenous substrate concentration, incubation time and cell number, thereby indicating that our method appropriately evaluates enzyme-mediated generation of HETEs. LTC4 generation after A23187 stimulation was also dependent on incubation time and cell number, but not on exogenous substrate concentration. Thus, LTC4 generation was mostly due to endogenous supply of the substrate from cellular membranes. Our present study indicates that lZHETEand LTC4-generating capacities are significantly in-
188 Prostaglandins Leukotrienes
and Essential Fatty Acids
“9 t 1.5x10* cells
INCUBATION TIME (min)
CELL NUMBER ngfh ng/h/4xlO'
cells
TjL!L__ 1.5
3.0
4.5
CELL NUMBER (x1o’ cel’s)
Fig. 2 Time, cell-number and exogenous-substrate dependencies of HETE and LTC, generations from macrophages. A,B: time courses, CD: cell number dependency and E,F: exogenous substrate dependency; A,C,E: for HETEs and B,D,F: for LTC,. The generation was examined using the same reaction method as in the legend of Figure 1, except for various reaction times in A B, various cell numbers in C,D or various arachidonate concentrations in E,F. HETE reduction is expressed as r&.5 x lo1 cells in A, ngih in C and ng/2 X IOr’cells /h in E, and LTC, production as ng/4 x 10? cells m B, n&t in D and n&/4 X lo6 cells in F.
DAYS
AFTER
TUMOR
INJECTION
Fig. 3 Effects of B16 melanoma cell-bearing conditions on HETE and LT generation capacities. B16 melanoma cells (2 X 10” cells) in saline were subcutaneously inoculated into the chest. Saline only was injected for O-day controls. At various days after tumor inoculation, peritoneal macrophages were obtained and analyzed in the same way as described in the legend of Figure 1. HETEs and LTs were quantitated by their absorbances using absorption coefficients of authentic standards. 12-HETE ressed as ngh/2 X 10 ceils; 15HETE and 5-HETE as X 10” cells and LTC, as t&r/8 x 10” ceils. Data are :he means + S.D. of five separate experiments. *: p < 0.05.
creased on the fourth day after the tumor inoculation, and return to the control level by the 11th day. Interestingly, such an increase in the early stage after tumor inoculation can also be seen in the generation capacity of cyclooxygenase products like PGE2 and 6-keto-PGF1, (11, 12). However, later time-courses appear to be different in that the enhanced capacity for the synthesis was transient for 1ZHETE and LTC4, but was persistent for the cyclooxygenase products (12). The enhanced PG production in tumor-bearing macrophages was explained by enhanced conversion efficiency of arachidonic acid into PGs (11). The mechanism or enzymes involved in the temporary increase in the generation capacity of 1ZHETE and LTC4 in the early stage were not elucidated in this study. 12-HETE has been reported to induce chemotaxis for neutrophils, degranulation of neutrophils, chemokinesis for smooth muscle cells and an increase in mononuclear procoagulant activity (13). Enhanced production of IZHETE after tumor inoculation was also demonstrated with rat alveolar
HETEs and LTs in Peritoneal Macrophages of Tumor-bearing
macrophages by Kort et al and Vincent et al (14, 15). Among lipoxygease products, LTB4 has been mostly mentioned in relation to cytokine production and tumor-growth inhibition (16-21). However, in mouse peritoneal macrophages the production of LTB4 appears to be negligible, if present at all, as compared with LTC4 which is produced in large amounts. LTC4 has been reported to induce interferon-y output from T-cells (16, 19), along with or thereby stimulating the release of interleukin 1 or 2 (8, 16, 18, 19) and tumor necrosis factor (8). Such cytokine production was enhanced by a cyclooxygenase inhibitor (20) while it was attenuated by lipoxygenase inhibitors (19). LTC4 also enhances macrophage cytostatic activity by the aid of increased cytosolic Ca*+ (22) or after metabolic conversion (21). The inhibition of 5lipoxygenase of peritoneal macrophages activated by A23187 was demonstrated to have the effect of inhibiting the cytotoxic activity towards tumor cells (23). Oxygen radicals produced by lipoxygenation have been related to natural killer cell-mediated cytotoxicity (24). Likewise, in macrophages it would be possible that lipoxygenation processes leading to LTC4, 12HETE or other lipoxygenase products release cytotoxic oxygen radicals. Theoretically, macrophages, natural killer T-cells and granulocytes migrating to tumor cells could exhibit anti-tumor suppressive activities. Thus, it appears that the generation of LTC4 and probably 12-HETE may bring about suppressive functions on tumor progression, in contrast with PGE?, which augments both tumor-mediated immunosuppression and tumor growth. The increase in lipoxygenase products shown in our present study may induce a positive immume response. However, this positive response may be masked by the immunosuppressive action of PGE?, produced in large amounts after tumor implantation. Acknowledgements We thank Ono Pharmaceutical Company (Osaka, Japan) for providing a generous supply of synthetic LTs, HETEs and PGBz. This work was partly supported by grants from the Ministry of Education, Science and Culture of Japan (No. 63570462)
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