0021-972x/92/7401-0130$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 74, No. 1 in U.S.A.
Printed
Tumor Necrosis Factor-a! Gonadotropin Secretion* KAZUTOMO OHASHIP, AKINORI WAKIMOTO,
Inhibits
FUMITAKA SAJI, MUNEHIRO OSAMU TANIZAWA
Human KATO,
AND
Department of Obstetrics and Gynecology, Osaka University Medical School, l-l-50 Fukushima-ku, Osaka 553, Japan
the direct
effects
of TNFa
on trophoblasts,
Fukushima,
h to 88%, 81%, and 71% of the control level, respectively. The expression of hCGB mRNA was also decreased to 23% of the control level after 24 h of TNFa treatment (100 U/mL). Inhibition occurred after 12 h of TNFa treatment (100 U/mL). Two measurements, trypan blue dye exclusion and 3-(4,5dimethylthiazol-2-v1)2.5-diuhenvl tetrazolium bromide reduction. revealed that these inhibitory effects were not due to the cytotoxic activity of TNFa on NUCl. In conclusion, TNFa reduces hCG secretion in uitro. (J Clin Endocrirwl Metub 74: 130-134, 1992)
ABSTRACT. The placenta is a major source of tumor necrosis factor-a (TNFa) and is rich in TNFa receptors. TNFa inhibited hCG secretion by normal chorionic villi at 6 weeks gestation, but chorionic villi contain the heterogenous cell population. To investigate
Chorionic
the NUCl
choriocarcinoma cell line was used as an in uitro placental cell model. TNFa also inhibited hCG secretion by NUCl cells at concentrations from l-100 U/mL. TNFa at concentrations of 1, 10, and 100 U/mL significantly decreased hCG secretion for 24
T
in the culture supernatant was determined by RIA, and the level of hCG mRNA expression was analyzed by Northern blot hybridization.
HE PLACENTA has been identified as a major source of some cytokines, including interleukin-1 (l), interleukin-6 (2), and tumor necrosis factor-a (TNFa) (3, 4). TNFa was originally found to be a mediator in the immunological system and has a wide variety of biological functions (5). It is produced by trophoblasts and decidual cells as an inflammatory mediator when stimulated by bacterial lipopolysaccharides (LPS) (1, 3). In addition, TNFa receptors have been detected on trophoblasts (6). Romero et al. (4) reported that TNFa stimulated prostaglandin production to induce labor and that TNFcx production resulting from maternal infection might trigger labor. However, the biological significance of TNFa during the early stage of pregnancy remains to be clarified. Recently, TNF~x has been reported to be a suppressive regulator in the endocrine system (7, 8). In this study we examined whether TNFa could modulate hCG secretion from human trophoblasts in early pregnancy. To elucidate the effects of TNFcx on hCG secretion, we used explants of chorionic villi and also studied the NUCl hCG-secreting choriocarcinoma cell line (9) as an in vitro model of trophoblasts. After exposure to TNFa, the hCG concentration
Materials Chorionic
and Methods
villous preparation
Normal chorions were obtained after therapeutic abortion at 6 weeks gestation. Chorionic villi were cleaned of maternal decidua and blood clots, rinsed repeatedly with phosphate buffered saline, and minced with scissors to isolate villous fragments at X10 magnification under a dissecting microscope. Culture
of
chorionic villous and NUCl cells
Chorionic villous and NUCl choriocarcinoma cells were cultured in RPMI-1640 medium containing 10% fetal calf serum (FCS; Hyclone, Logan, UT), streptomycin (100 pg/mL), and penicillin (100 U/mL) at 37 C in 5% CO, in humidified air. Approximately 10 mg wet weight villous explants and 4 x 10” NUCl cells in 1-mL aliquots were placed in 12-well tissue culture plates (Corning, Corning, NY). Explants and cells were incubated in the presence of TNFa at various concentrations. At the end of incubation, villous explants, NUCl cells, and the respective conditioned culture media were harvested.
Received December 31, 1990. * This work was supported by grants-in-aid for scientific research (no. 01570929 and 02454383) from the Ministry of Education, Science, and Culture of Japan. t To whom all correspondence and requests for reprints should be addressed.
Assessment of cell viability Cell viability was determined by trypan blue dye exclusion and the extent of reduction of the chromogen 3-(4,5dimethylthiazol-2-y1)2,5diphenyl tetrazolium bromide (MTT; Sigma, 130
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 10:49 For personal use only. No other uses without permission. . All rights reserved.
TNF INHIBITION St. Louis, MO). Trypan blue dye exclusion was performed at the completion of experiments. Cells were incubated for 5 min in 0.08% trypan blue-RPMI-1640, and their numbers were counted. We assessedthe extent of MTT reduction in a separate experiment. NUCl cells were suspended in RPMI-164010% FCS in the presence or absence of TNFLu, and 1 X lo4 cells in lOO-rL aliquots were cultured in 96-well tissue culture plates (Corning). After 24 h of incubation, 10 PL RPMI-1640 containing 6 mg/ml MTT were added to each well, and the cells were incubated for a further 4 h. The culture medium was then aspirated, the cells were lysed with 100 PL 0.04 N hydrochloric acid in isopropanol, and an equal volume of distilled water was added to each well. MTT reduction was determined by reading the plates at the optical density of 570/690 nm on an automated enzyme-linked immunosorbant assay plate reader (Corona Electric, Tokyo, Japan). TNFol
Human recombinant TNFa was a kind gift from Dainihon Pharmaceutical Co. (Osaka, Japan). It has been reported to exert cytostatic and cytocidal effects against a broad spectrum of human tumor cells, and its cytotoxic activity is tumor specific (10). TNFa was stored at 4 C and was diluted immediately before use (1 U = 0.33 ng protein). hCG, µglobulin measurements
c&m), and cellular protein
We used a time-resolved fluoroimmunoassay for hCG with monoclonal antibodies directed against the LY-and P-subunits (DELFIA hCG kit, Pharmacia, Uppsala, Sweden). The hCG detected by this assay was the mature hCG dimer consisting of (Y-and p-subunits. The ,&m level in the culture supernatant was measured with a /3:! MICRO-RIA kit (Pharmacia). Chorionic villi were washed with phosphate-buffered saline, solubilized in 1 N NaOH for 12 h at room temperature, and then neutralized with 1 N HCl. The protein content was determined by the Lowry reaction. cDNA probes
The 540-base pair cDNA of the hCG P-subunit was a generous gift from Dr. Hayashizaki (Osaka University). The cDNA fragment encoding almost the full-length mRNA for the psubunit was cut out at the Hind111 sites. The &m probe, a PstI fragment (545 basepairs) of the human finrn cloned in pBR322, was kindly provided by Dr. Itakura (City of Hope Research Institute, Duarte, CA) (11). Extraction
of
total RNA and Northern
blot analysis
Total RNA was extracted from choriocarcinoma cells by homogenization in the presence of 4 M guanidine thiocyanate, centrifugation through a cushion of 5.7 M cesium chloride, and phenol precipitation. Ten micrograms of each RNA sample in 6 M glyoxal-dimethylsulfoxide were denatured and subjected to electrophoresis on 1% agarose gel. RNA was then transferred to a nylon membrane and hybridized with the ““P-labeled hCGP or &rn probe. The filters were washed several times at room
OF hCG SECRETION
131
temperature and exposed to AIF-RX film (Fuji Photo Film, Tokyo, Japan) at -70 C with an intensifying screen. Hybridizing band intensities were determined by a scanning densitometer (Bio-Rad, Richmond, CA). Statistical
analysis
Experimental data are presented as the mean + SD. The data were analyzed by one-way analysis of variance, followed by Dunnett’s multiple range test and Student’s t test.
Results We first determined whether TNFcv affected hCG secretion from trophoblasts in early pregnancy. Without enzyme treatment, chorionic villous fragments were isolated from chorions obtained after therapeutic abortion at 6 weeks gestation. Explants of the chorionic villi in RPMI-1640-10% FCS secreted 30.2 + 2.5 X lo3 and 25.0 + 1.9 X lo3 IU/mg protein in 24 h. TNFa! significantly decreased hCG secretion compared to that in control medium at both 10 and 100 U/mL (Table 1). Chorionic villi contain trophoblasts, placental macrophages, and decidual cells, which produce several humoral factors and have various influences on each other. To investigate the direct effects of TNFa on trophoblasts, NUCl choriocarcinoma cells were used as an in vitro trophoblast model. NUCl cells in the absence of TNFa secreted 285.0 f 20.3 IU/L hCG in 24 h. As shown in Table 2, TNFcx decreased hCG secretion by NUCl cells. After treatment with 1, 10, and 100 U/ml TNFa for 24 h, the hCG concentrations in the culture supernatant were 88%, 81%, and 71% of the control value, respectively, indicating that TNFcr significantly reduced hCG secretion (P < 0.01). Since TNFa is toxic to some tumor cells, we examined the possibility that the inhibition of hCG secretion was due to the reduction of cellular proliferation. Proliferation was assessed by two methods, TABLE 1. hCG in culture supernatant of chorionic villi after TNFa treatment for 24 h
hCG secretion(X10” IU/mg protein)” TNFa W/mL)
Exp 1 Mean +
0 1 10 100
30.2 27.6 25.1 24.2
+ + + +
SD
2.5 2.3 2.3 3.4
Exp 2 Range
27.8-34.4
24.1-29.8 21.3-27.2* 20.4-28.4’
Mean & 25.0 23.7 21.8 21.3
+ f f +
SD
2.1 2.8 1.6 2.1
Range 22.6-27.9 21.1-27.5 20.0-23.8* 18.6-23.5’
Chorionic villus explants were cultured in the absenceor presence of TNFa at different conditions. hCG levelswere determined in medium collectedafter 24 h of TNFa treatment. ’ n = 5 for all groups. ’ Significantly different from control (P < 0.05), by Dunnett’s multiple range test. ’ Significantly different from control (P < O.Ol),by Dunnett’s multiple range test.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 10:49 For personal use only. No other uses without permission. . All rights reserved.
OHASHI
132 TABLE 2. hCG and treatment for 24 h
PLrn in culture
hCG
TNFa UJ/mL)
0 1 10 100
Mean 285.0 251.0 231.0 201.8
supernatant
secretion
TNFa
0 hCG-B
Pm (rgl mL)h
Range 262.8-319.1 228.9-271.4 208.0-244.1 184.6-235.8
20.3 16.7’ 15.6’ 17.7’
after
0.2 0.2 0.2 0.3
+ + + r
TNFa W/mL)
0 1 10 100
and viability
Trypan blue dye exclusion” Dead cell (%)
5.3 5.6 5.2 5.4
4.4 6.5 6.4 4.3
0.3 0.3 0.3 0.2
rt f + f
after
MTT
Cell count (X101) + f + +
of NUCl
0.1 0.1 0.1 0.1
0.9 2.7 2.3 1.1
TNFor
hCG+ b
treat-
reduction*
%
Range
100 99 f 17 100 + 19 96 + 17
79-118 73-116 82-123
Cell proliferation and viability were determined by trypan exclusion and MTT reduction, as described in the text. ” n = 3 for all groups. ’ n = 5 for all groups.
blue
0 pz m
(a)
Cells were cultured in the absence or presence of TNFa at different concentrations. hCG levels were determined in medium collected after 24 h of TNFol treatment. ” n = 6 for all groups. ” n = 3 for all groups. Values are the mean + SD. ’ Significantly different from control (P < O.Ol), by Dunnett’s multiple range test. TABLE 3. Cell proliferation ment for 24 h
JCE& M-1992 Voll4.Nol
(cl
(W/L)”
+ SD + k + +
of NUCl
ET AL.
(b)
1
p.2 m ’
TNFa
(U/ml)
FIG. 1. Northern blot hybridization analysis of RNA hybridized to hCG@ cDNA. Cells were incubated for 24 h in the presence of TNFa at different concentrations. RNA was extracted and analyzed, as described in Materials and Methods. The probes were: a, hCGp; and b, P1rn. c, Levels of hCG@ mRNA in NUCl in response to TNFL~ treatment. Values plotted are from a densitometric scan of the radioautograms and show the relative amounts of hCGfl (0) and &rn (0).
250
(P
Vol. 74, No. 1 in U.S.A.
Printed
Tumor Necrosis Factor-a! Gonadotropin Secretion* KAZUTOMO OHASHIP, AKINORI WAKIMOTO,
Inhibits
FUMITAKA SAJI, MUNEHIRO OSAMU TANIZAWA
Human KATO,
AND
Department of Obstetrics and Gynecology, Osaka University Medical School, l-l-50 Fukushima-ku, Osaka 553, Japan
the direct
effects
of TNFa
on trophoblasts,
Fukushima,
h to 88%, 81%, and 71% of the control level, respectively. The expression of hCGB mRNA was also decreased to 23% of the control level after 24 h of TNFa treatment (100 U/mL). Inhibition occurred after 12 h of TNFa treatment (100 U/mL). Two measurements, trypan blue dye exclusion and 3-(4,5dimethylthiazol-2-v1)2.5-diuhenvl tetrazolium bromide reduction. revealed that these inhibitory effects were not due to the cytotoxic activity of TNFa on NUCl. In conclusion, TNFa reduces hCG secretion in uitro. (J Clin Endocrirwl Metub 74: 130-134, 1992)
ABSTRACT. The placenta is a major source of tumor necrosis factor-a (TNFa) and is rich in TNFa receptors. TNFa inhibited hCG secretion by normal chorionic villi at 6 weeks gestation, but chorionic villi contain the heterogenous cell population. To investigate
Chorionic
the NUCl
choriocarcinoma cell line was used as an in uitro placental cell model. TNFa also inhibited hCG secretion by NUCl cells at concentrations from l-100 U/mL. TNFa at concentrations of 1, 10, and 100 U/mL significantly decreased hCG secretion for 24
T
in the culture supernatant was determined by RIA, and the level of hCG mRNA expression was analyzed by Northern blot hybridization.
HE PLACENTA has been identified as a major source of some cytokines, including interleukin-1 (l), interleukin-6 (2), and tumor necrosis factor-a (TNFa) (3, 4). TNFa was originally found to be a mediator in the immunological system and has a wide variety of biological functions (5). It is produced by trophoblasts and decidual cells as an inflammatory mediator when stimulated by bacterial lipopolysaccharides (LPS) (1, 3). In addition, TNFa receptors have been detected on trophoblasts (6). Romero et al. (4) reported that TNFa stimulated prostaglandin production to induce labor and that TNFcx production resulting from maternal infection might trigger labor. However, the biological significance of TNFa during the early stage of pregnancy remains to be clarified. Recently, TNF~x has been reported to be a suppressive regulator in the endocrine system (7, 8). In this study we examined whether TNFa could modulate hCG secretion from human trophoblasts in early pregnancy. To elucidate the effects of TNFcx on hCG secretion, we used explants of chorionic villi and also studied the NUCl hCG-secreting choriocarcinoma cell line (9) as an in vitro model of trophoblasts. After exposure to TNFa, the hCG concentration
Materials Chorionic
and Methods
villous preparation
Normal chorions were obtained after therapeutic abortion at 6 weeks gestation. Chorionic villi were cleaned of maternal decidua and blood clots, rinsed repeatedly with phosphate buffered saline, and minced with scissors to isolate villous fragments at X10 magnification under a dissecting microscope. Culture
of
chorionic villous and NUCl cells
Chorionic villous and NUCl choriocarcinoma cells were cultured in RPMI-1640 medium containing 10% fetal calf serum (FCS; Hyclone, Logan, UT), streptomycin (100 pg/mL), and penicillin (100 U/mL) at 37 C in 5% CO, in humidified air. Approximately 10 mg wet weight villous explants and 4 x 10” NUCl cells in 1-mL aliquots were placed in 12-well tissue culture plates (Corning, Corning, NY). Explants and cells were incubated in the presence of TNFa at various concentrations. At the end of incubation, villous explants, NUCl cells, and the respective conditioned culture media were harvested.
Received December 31, 1990. * This work was supported by grants-in-aid for scientific research (no. 01570929 and 02454383) from the Ministry of Education, Science, and Culture of Japan. t To whom all correspondence and requests for reprints should be addressed.
Assessment of cell viability Cell viability was determined by trypan blue dye exclusion and the extent of reduction of the chromogen 3-(4,5dimethylthiazol-2-y1)2,5diphenyl tetrazolium bromide (MTT; Sigma, 130
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 10:49 For personal use only. No other uses without permission. . All rights reserved.
TNF INHIBITION St. Louis, MO). Trypan blue dye exclusion was performed at the completion of experiments. Cells were incubated for 5 min in 0.08% trypan blue-RPMI-1640, and their numbers were counted. We assessedthe extent of MTT reduction in a separate experiment. NUCl cells were suspended in RPMI-164010% FCS in the presence or absence of TNFLu, and 1 X lo4 cells in lOO-rL aliquots were cultured in 96-well tissue culture plates (Corning). After 24 h of incubation, 10 PL RPMI-1640 containing 6 mg/ml MTT were added to each well, and the cells were incubated for a further 4 h. The culture medium was then aspirated, the cells were lysed with 100 PL 0.04 N hydrochloric acid in isopropanol, and an equal volume of distilled water was added to each well. MTT reduction was determined by reading the plates at the optical density of 570/690 nm on an automated enzyme-linked immunosorbant assay plate reader (Corona Electric, Tokyo, Japan). TNFol
Human recombinant TNFa was a kind gift from Dainihon Pharmaceutical Co. (Osaka, Japan). It has been reported to exert cytostatic and cytocidal effects against a broad spectrum of human tumor cells, and its cytotoxic activity is tumor specific (10). TNFa was stored at 4 C and was diluted immediately before use (1 U = 0.33 ng protein). hCG, µglobulin measurements
c&m), and cellular protein
We used a time-resolved fluoroimmunoassay for hCG with monoclonal antibodies directed against the LY-and P-subunits (DELFIA hCG kit, Pharmacia, Uppsala, Sweden). The hCG detected by this assay was the mature hCG dimer consisting of (Y-and p-subunits. The ,&m level in the culture supernatant was measured with a /3:! MICRO-RIA kit (Pharmacia). Chorionic villi were washed with phosphate-buffered saline, solubilized in 1 N NaOH for 12 h at room temperature, and then neutralized with 1 N HCl. The protein content was determined by the Lowry reaction. cDNA probes
The 540-base pair cDNA of the hCG P-subunit was a generous gift from Dr. Hayashizaki (Osaka University). The cDNA fragment encoding almost the full-length mRNA for the psubunit was cut out at the Hind111 sites. The &m probe, a PstI fragment (545 basepairs) of the human finrn cloned in pBR322, was kindly provided by Dr. Itakura (City of Hope Research Institute, Duarte, CA) (11). Extraction
of
total RNA and Northern
blot analysis
Total RNA was extracted from choriocarcinoma cells by homogenization in the presence of 4 M guanidine thiocyanate, centrifugation through a cushion of 5.7 M cesium chloride, and phenol precipitation. Ten micrograms of each RNA sample in 6 M glyoxal-dimethylsulfoxide were denatured and subjected to electrophoresis on 1% agarose gel. RNA was then transferred to a nylon membrane and hybridized with the ““P-labeled hCGP or &rn probe. The filters were washed several times at room
OF hCG SECRETION
131
temperature and exposed to AIF-RX film (Fuji Photo Film, Tokyo, Japan) at -70 C with an intensifying screen. Hybridizing band intensities were determined by a scanning densitometer (Bio-Rad, Richmond, CA). Statistical
analysis
Experimental data are presented as the mean + SD. The data were analyzed by one-way analysis of variance, followed by Dunnett’s multiple range test and Student’s t test.
Results We first determined whether TNFcv affected hCG secretion from trophoblasts in early pregnancy. Without enzyme treatment, chorionic villous fragments were isolated from chorions obtained after therapeutic abortion at 6 weeks gestation. Explants of the chorionic villi in RPMI-1640-10% FCS secreted 30.2 + 2.5 X lo3 and 25.0 + 1.9 X lo3 IU/mg protein in 24 h. TNFa! significantly decreased hCG secretion compared to that in control medium at both 10 and 100 U/mL (Table 1). Chorionic villi contain trophoblasts, placental macrophages, and decidual cells, which produce several humoral factors and have various influences on each other. To investigate the direct effects of TNFa on trophoblasts, NUCl choriocarcinoma cells were used as an in vitro trophoblast model. NUCl cells in the absence of TNFa secreted 285.0 f 20.3 IU/L hCG in 24 h. As shown in Table 2, TNFcx decreased hCG secretion by NUCl cells. After treatment with 1, 10, and 100 U/ml TNFa for 24 h, the hCG concentrations in the culture supernatant were 88%, 81%, and 71% of the control value, respectively, indicating that TNFcr significantly reduced hCG secretion (P < 0.01). Since TNFa is toxic to some tumor cells, we examined the possibility that the inhibition of hCG secretion was due to the reduction of cellular proliferation. Proliferation was assessed by two methods, TABLE 1. hCG in culture supernatant of chorionic villi after TNFa treatment for 24 h
hCG secretion(X10” IU/mg protein)” TNFa W/mL)
Exp 1 Mean +
0 1 10 100
30.2 27.6 25.1 24.2
+ + + +
SD
2.5 2.3 2.3 3.4
Exp 2 Range
27.8-34.4
24.1-29.8 21.3-27.2* 20.4-28.4’
Mean & 25.0 23.7 21.8 21.3
+ f f +
SD
2.1 2.8 1.6 2.1
Range 22.6-27.9 21.1-27.5 20.0-23.8* 18.6-23.5’
Chorionic villus explants were cultured in the absenceor presence of TNFa at different conditions. hCG levelswere determined in medium collectedafter 24 h of TNFa treatment. ’ n = 5 for all groups. ’ Significantly different from control (P < 0.05), by Dunnett’s multiple range test. ’ Significantly different from control (P < O.Ol),by Dunnett’s multiple range test.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 10:49 For personal use only. No other uses without permission. . All rights reserved.
OHASHI
132 TABLE 2. hCG and treatment for 24 h
PLrn in culture
hCG
TNFa UJ/mL)
0 1 10 100
Mean 285.0 251.0 231.0 201.8
supernatant
secretion
TNFa
0 hCG-B
Pm (rgl mL)h
Range 262.8-319.1 228.9-271.4 208.0-244.1 184.6-235.8
20.3 16.7’ 15.6’ 17.7’
after
0.2 0.2 0.2 0.3
+ + + r
TNFa W/mL)
0 1 10 100
and viability
Trypan blue dye exclusion” Dead cell (%)
5.3 5.6 5.2 5.4
4.4 6.5 6.4 4.3
0.3 0.3 0.3 0.2
rt f + f
after
MTT
Cell count (X101) + f + +
of NUCl
0.1 0.1 0.1 0.1
0.9 2.7 2.3 1.1
TNFor
hCG+ b
treat-
reduction*
%
Range
100 99 f 17 100 + 19 96 + 17
79-118 73-116 82-123
Cell proliferation and viability were determined by trypan exclusion and MTT reduction, as described in the text. ” n = 3 for all groups. ’ n = 5 for all groups.
blue
0 pz m
(a)
Cells were cultured in the absence or presence of TNFa at different concentrations. hCG levels were determined in medium collected after 24 h of TNFol treatment. ” n = 6 for all groups. ” n = 3 for all groups. Values are the mean + SD. ’ Significantly different from control (P < O.Ol), by Dunnett’s multiple range test. TABLE 3. Cell proliferation ment for 24 h
JCE& M-1992 Voll4.Nol
(cl
(W/L)”
+ SD + k + +
of NUCl
ET AL.
(b)
1
p.2 m ’
TNFa
(U/ml)
FIG. 1. Northern blot hybridization analysis of RNA hybridized to hCG@ cDNA. Cells were incubated for 24 h in the presence of TNFa at different concentrations. RNA was extracted and analyzed, as described in Materials and Methods. The probes were: a, hCGp; and b, P1rn. c, Levels of hCG@ mRNA in NUCl in response to TNFL~ treatment. Values plotted are from a densitometric scan of the radioautograms and show the relative amounts of hCGfl (0) and &rn (0).
250
(P
