0021-972X/91/7306-1170$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society
Vol. 73, No. 6 Printed in U.S.A.
Interleukin-1 Inhibits in Vitro Decidualization of Human Endometrial Stromal Cells* MASATOSHI KARIYA, HIDEHARU KANZAKI, KENJI TAKAKURA, KIMITOSHI IMAI, NORIHIKO OKAMOTO, NOBUYUKI EMI, YOSHITAKA KARIYA, AND TAKAHIDE MORI Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
1. IL-1 markedly suppressed the induction of PRL production by P in a dose-dependent manner. The morphological decidualization of ESC in response to P was also inhibited by IL-1. This report demonstrates for the first time the possibility that IL-1 blocks decidualization, the functional differentiation of human endometrial stromal cells in response to ovarian steroids. (J Clin Endocrinol Metab 73: 1170-1174, 1991)
ABSTRACT. Interleukin-1 (IL-1), a critical cytokine for the initiation of the immune response to infection or antigenic challenge, is known to also possess a variety of biological functions outside the immune system. We examined whether IL-1 could affect the decidualization of human endometrial stromal cells (ESC), a conspicuous part in the process of implantation, by assessing PRL production and morphological transformation in an in vitro system. Purified human ESC were cultured in the presence of progesterone (P) with or without the addition of IL-
T
IL-1 can affect reproductive functions; the inhibition of LH-induced luteinization of porcine granulosa cells (8), the stimulation of hCG secretion of human trophoblast (9), the induction of IL-6 secretion by human endometrial stromal cells (10), and the stimulation of PGE2 production by human endometrial epithelium (11). In the present study we established an in vitro model of decidualization by culturing human ESC under the influence of progesterone (P) according to the method previously reported (12-14) and examined the effect of IL-1 on decidualization, as assessed by PRL secretion and morphological changes.
O ACHIEVE successful implantation, timely changes in endometrium, secretory changes in glandular epithelium, and subsequent decidual transformation of stromal cells (decidualization) are thought to be very important. Decidualization is known to be regulated mainly by ovarian hormones released by the corpus luteum, but our knowledge of the details of its regulation is limited, especially with regard to the decidualization of human endometrial stromal cells (ESC). In other species, it has become apparent that factors other than ovarian hormones can modulate decidualization, including prostaglandins (1) and leukotrienes (2). Interleukin-1 (IL-1), a cytokine released predominantly by activated macrophages and monocytes after infection, injury, or antigenic challenge, activates lymphocytes and plays an important role in the initiation of the immune response (3). IL-1 has also been reported to possess a variety of other biological activities. For example, it stimulates proliferation and collagen synthesis of fibroblasts (4, 5), and augments prostaglandin E2 (PGE2) synthesis by several types of cells (6, 7). In addition, it has recently been demonstrated in vitro that
Materials and Methods
Received August 28,1990. Address all correspondence and requests for reprints to: Masatoshi Kariya, M.D., Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan. * This work was supported in part by Grants-in-Aid for Scientific Research (no. 02222104 and 01570928) from the Ministry of Education, Sciences, and Culture of Japan.
Human endometrium was obtained at hysterectomy from five normally cycling premenopausal women, aged 38-48 yr, who underwent surgery for myoma uteri. A portion of each endometrial specimen obtained was examined histologically and dated according to the criteria of Noyes et al. (15). Three specimens were diagnosed as late proliferative, and the other two were midsecretory (day 23). Tissue samples were washed with RPMI-1640 (Gibco, Grand Island, NY) and minced into small pieces of less than 1 mm3. Then, the tissues were incubated for 2 h at 37 C in RPMI-1640 containing 0.2% collagenase (Wako Co. Ltd., Osaka, Japan) and 0.005% deoxyribonuclease (Sigma Chemical Co., St. Louis, MO). After this enzymatic digestion, cell clumps were dispersed by pipetting. Most of the stromal cells were then present as single cells or small aggregates, while most of the epithelial cells remained in larger clumps. Stromal cells were separated
1170
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 22:37 For personal use only. No other uses without permission. . All rights reserved.
IL-1 INHIBITS DECIDUALIZATION by differential sedimentation at unit gravity, as follows. The cell suspension was placed in a 15-mL polyethylene centrifugation tube (Corning Glass Works, Corning, NY) and left in a upright position for 5-10 min. During this procedure, cell aggregates or clumps precipitate onto the bottom. Then, the supernatant was transferred into a new tube to collect suspending cells. After repeating this procedure two or three times, stromal cells of more than 90% purity were obtained. These cells were washed three times, and the number of viable cells was counted by dye exclusion using erythrocin-B. The viability of separated stromal cells was at least 90% in each experiment. Two million viable cells were inoculated into each of the wells of six-well plates (Beckton Dickinson Co., Rutherford, NJ). Cells were cultured in triplicate with 4 mL RPMI-1640 supplemented with 10% fetal calf serum (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan), 100 IU/mL penicillin, 100 \igl mL streptomycin, and 100 nmol/L P (4-pregnene-3,20-dione; Sigma) for 12-16 days at 37 C in a humidified atmosphere of 5% CO2 in air. Recombinant human IL-la (2 X 107 U/mg protein; Dainippon Pharmaceutical Co. Ltd.) was added to the culture from the initiation, and the culture medium was changed every 2 days, with supplementation by IL-1 and P. At the completion of culture, culture media were collected, centrifuged, and frozen at -20 C until the PRL assay was performed. The number of cultured cells was also counted by the citric acid-crystal violet method. PRL concentration was determined by RIA using a commercial kit (Daiichi Radioisotope Lab. Ltd., Tokyo, Japan). The detection limit was 1.0 ng/ L, and the intra- and interassay coefficients of variation were 1.9-7.1% and 1.6-3.6%, respectively. Each PRL assay was performed in duplicate. PRL values were standardized on the basis of the cell number counted at the time culture media were collected for PRL assay, and data were analyzed statistically using the unpaired t test. For morphological assessment, part of each culture was stained with Giemsa stain and observed by phase contrast microscopy. In Exp 1, the IL-1 effect on ESC proliferation was assessed by the [3H]thymidine incorporation into cultured cells as well as by cell numbers. Ten thousand ESC were inoculated in each well of a 96-well plate (Corning Glass Works) and cultured in the presence or absence of IL-1 in RPMI-1640 medium containing 10% fetal calf serum and antibiotics for 3 days at 37 C in a humidified atmosphere of 5% CO2 in air in a total volume of 0.2 mL in quadruplicate. ESC were labeled with [3H]thymidine (2 Ci/mmol; Amersham, Aylesburg, Buckinghamshire, United Kingdom), adding 0.5 ixCi to each well for last 12 h of the culture. At the end of the culture, ESC were harvested on filter papers, and the radioactivity was counted in a liquid scintillation counter (Aloka, Co., Tokyo, Japan).
Results In the presence of P alone, PRL was first detectable in culture supernatants after 2-4 days of lag period, and subsequently, the level increased steeply to reach 10-50 ng/day-106 cells at the completion of culture. In the absence of P, however, PRL could not be detected in the ESC culture supernatants of every five cases after 12-16
1171
days of culture. The addition of IL-1 (2 U/mL) markedly inhibited the P-dependent induction of PRL secretion by stromal cells in all five experiments (Table 1). In Exp 1, the inhibitory effect of IL-1 on PRL production was dose dependent, without causing a significant change in the cell numbers up to a concentration of 2 U/mL (Fig. 1). There was also no difference in cell numbers between stromal cells cultured with and without IL-1 after 12-16 days of culture in the other four experiments (data not shown). In Exp 1, [3H]thymidine uptake of stromal cells cultured in the absence or presence of IL-1 also showed no significant differences (Table 2). Morphological studies showed that spindle-shaped stromal cells (at the start of culture; Fig. 2A) were transformed in the presence of P into large polygonal cells, resembling decidual cells in vivo (Fig. 2B). In contrast, stromal cells cultured with P plus IL-1 remained fibroblastic in appearance even at the completion of culture (Fig. 2C).
Discussion Decidualization is known to be under the control of ovarian steroid hormones. In addition, it has become apparent in various mammalian species that some other factors act as local mediators in the regulation of decidualization, including PGs (1), leukotrienes (2), and platelet-activating factor (16). In humans, however, the existence of local mediators modulating this process has not previously been proven. The present study suggests that IL-1, one of the key mediators of immunological reaction predominantly released from activated macrophages, can work as a local modulator in the decidualization of human endometrium. It has been reported that PRL is produced by the late secretary endometrium of normally cycling women, and that its production correlates with the degree of histological decidualization (17). It has also been reported that PRL is not detected in ESC by immunohistochemistry until they are decidualized (18). Morphologically, decidualization is characterized by the conversion of spindleshaped stromal cells into epithelial-like cells with enlarged nuclei and an increased amount of cytoplasm (19). ESC cultured in the presence of P produced PRL and were transformed into large epithelial-like cells similar to those of in vivo decidua. Irwin et al. (12) have reported that ESC cultured under the influence of ovarian steroid hormones show the ultrastructural and biochemical changes characteristic of decidualization in vivo. These biochemical changes are the production of laminin, fibronectin, and PRL (12). These similarities between Pinduced in vitro changes and in vivo decidualization suggest that this culture system is useful as an in vitro model for decidualization, and that PRL production and the morphological changes detectable in this culture
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 22:37 For personal use only. No other uses without permission. . All rights reserved.
KARIYA ET AL.
1172
JCE & M • 1991 Vol 73 • No 6
TABLE 1. Inhibitory effects of IL-1 on P-dependent PRL production by ESC PRL production (ng/day-106 cells)
Reagentsi P (nmol/L)
IL-1 (U/mL)
Expl, proliferative
Exp2, proliferative
Exp3, proliferative
100 100 100 100 100 100
0 0.002 0.02 0.2 2 20
42.2 ± 2.20 36.1 ± 0.15° 32.6 ± 0.986 26.2 ± 2.18* 11.3 ± 1.15* 11.2 ± 0.856
49.8 ± 1.79 ND ND ND 1.5 ± 0.25* 1.7 ± 0.286
8.6 ± 0.26 ND ND ND 2.9 ± 0.11" ND
Exp4, secretory 31.4 ± 5.38 ND ND ND
Vol. 73, No. 6 Printed in U.S.A.
Interleukin-1 Inhibits in Vitro Decidualization of Human Endometrial Stromal Cells* MASATOSHI KARIYA, HIDEHARU KANZAKI, KENJI TAKAKURA, KIMITOSHI IMAI, NORIHIKO OKAMOTO, NOBUYUKI EMI, YOSHITAKA KARIYA, AND TAKAHIDE MORI Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
1. IL-1 markedly suppressed the induction of PRL production by P in a dose-dependent manner. The morphological decidualization of ESC in response to P was also inhibited by IL-1. This report demonstrates for the first time the possibility that IL-1 blocks decidualization, the functional differentiation of human endometrial stromal cells in response to ovarian steroids. (J Clin Endocrinol Metab 73: 1170-1174, 1991)
ABSTRACT. Interleukin-1 (IL-1), a critical cytokine for the initiation of the immune response to infection or antigenic challenge, is known to also possess a variety of biological functions outside the immune system. We examined whether IL-1 could affect the decidualization of human endometrial stromal cells (ESC), a conspicuous part in the process of implantation, by assessing PRL production and morphological transformation in an in vitro system. Purified human ESC were cultured in the presence of progesterone (P) with or without the addition of IL-
T
IL-1 can affect reproductive functions; the inhibition of LH-induced luteinization of porcine granulosa cells (8), the stimulation of hCG secretion of human trophoblast (9), the induction of IL-6 secretion by human endometrial stromal cells (10), and the stimulation of PGE2 production by human endometrial epithelium (11). In the present study we established an in vitro model of decidualization by culturing human ESC under the influence of progesterone (P) according to the method previously reported (12-14) and examined the effect of IL-1 on decidualization, as assessed by PRL secretion and morphological changes.
O ACHIEVE successful implantation, timely changes in endometrium, secretory changes in glandular epithelium, and subsequent decidual transformation of stromal cells (decidualization) are thought to be very important. Decidualization is known to be regulated mainly by ovarian hormones released by the corpus luteum, but our knowledge of the details of its regulation is limited, especially with regard to the decidualization of human endometrial stromal cells (ESC). In other species, it has become apparent that factors other than ovarian hormones can modulate decidualization, including prostaglandins (1) and leukotrienes (2). Interleukin-1 (IL-1), a cytokine released predominantly by activated macrophages and monocytes after infection, injury, or antigenic challenge, activates lymphocytes and plays an important role in the initiation of the immune response (3). IL-1 has also been reported to possess a variety of other biological activities. For example, it stimulates proliferation and collagen synthesis of fibroblasts (4, 5), and augments prostaglandin E2 (PGE2) synthesis by several types of cells (6, 7). In addition, it has recently been demonstrated in vitro that
Materials and Methods
Received August 28,1990. Address all correspondence and requests for reprints to: Masatoshi Kariya, M.D., Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan. * This work was supported in part by Grants-in-Aid for Scientific Research (no. 02222104 and 01570928) from the Ministry of Education, Sciences, and Culture of Japan.
Human endometrium was obtained at hysterectomy from five normally cycling premenopausal women, aged 38-48 yr, who underwent surgery for myoma uteri. A portion of each endometrial specimen obtained was examined histologically and dated according to the criteria of Noyes et al. (15). Three specimens were diagnosed as late proliferative, and the other two were midsecretory (day 23). Tissue samples were washed with RPMI-1640 (Gibco, Grand Island, NY) and minced into small pieces of less than 1 mm3. Then, the tissues were incubated for 2 h at 37 C in RPMI-1640 containing 0.2% collagenase (Wako Co. Ltd., Osaka, Japan) and 0.005% deoxyribonuclease (Sigma Chemical Co., St. Louis, MO). After this enzymatic digestion, cell clumps were dispersed by pipetting. Most of the stromal cells were then present as single cells or small aggregates, while most of the epithelial cells remained in larger clumps. Stromal cells were separated
1170
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 22:37 For personal use only. No other uses without permission. . All rights reserved.
IL-1 INHIBITS DECIDUALIZATION by differential sedimentation at unit gravity, as follows. The cell suspension was placed in a 15-mL polyethylene centrifugation tube (Corning Glass Works, Corning, NY) and left in a upright position for 5-10 min. During this procedure, cell aggregates or clumps precipitate onto the bottom. Then, the supernatant was transferred into a new tube to collect suspending cells. After repeating this procedure two or three times, stromal cells of more than 90% purity were obtained. These cells were washed three times, and the number of viable cells was counted by dye exclusion using erythrocin-B. The viability of separated stromal cells was at least 90% in each experiment. Two million viable cells were inoculated into each of the wells of six-well plates (Beckton Dickinson Co., Rutherford, NJ). Cells were cultured in triplicate with 4 mL RPMI-1640 supplemented with 10% fetal calf serum (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan), 100 IU/mL penicillin, 100 \igl mL streptomycin, and 100 nmol/L P (4-pregnene-3,20-dione; Sigma) for 12-16 days at 37 C in a humidified atmosphere of 5% CO2 in air. Recombinant human IL-la (2 X 107 U/mg protein; Dainippon Pharmaceutical Co. Ltd.) was added to the culture from the initiation, and the culture medium was changed every 2 days, with supplementation by IL-1 and P. At the completion of culture, culture media were collected, centrifuged, and frozen at -20 C until the PRL assay was performed. The number of cultured cells was also counted by the citric acid-crystal violet method. PRL concentration was determined by RIA using a commercial kit (Daiichi Radioisotope Lab. Ltd., Tokyo, Japan). The detection limit was 1.0 ng/ L, and the intra- and interassay coefficients of variation were 1.9-7.1% and 1.6-3.6%, respectively. Each PRL assay was performed in duplicate. PRL values were standardized on the basis of the cell number counted at the time culture media were collected for PRL assay, and data were analyzed statistically using the unpaired t test. For morphological assessment, part of each culture was stained with Giemsa stain and observed by phase contrast microscopy. In Exp 1, the IL-1 effect on ESC proliferation was assessed by the [3H]thymidine incorporation into cultured cells as well as by cell numbers. Ten thousand ESC were inoculated in each well of a 96-well plate (Corning Glass Works) and cultured in the presence or absence of IL-1 in RPMI-1640 medium containing 10% fetal calf serum and antibiotics for 3 days at 37 C in a humidified atmosphere of 5% CO2 in air in a total volume of 0.2 mL in quadruplicate. ESC were labeled with [3H]thymidine (2 Ci/mmol; Amersham, Aylesburg, Buckinghamshire, United Kingdom), adding 0.5 ixCi to each well for last 12 h of the culture. At the end of the culture, ESC were harvested on filter papers, and the radioactivity was counted in a liquid scintillation counter (Aloka, Co., Tokyo, Japan).
Results In the presence of P alone, PRL was first detectable in culture supernatants after 2-4 days of lag period, and subsequently, the level increased steeply to reach 10-50 ng/day-106 cells at the completion of culture. In the absence of P, however, PRL could not be detected in the ESC culture supernatants of every five cases after 12-16
1171
days of culture. The addition of IL-1 (2 U/mL) markedly inhibited the P-dependent induction of PRL secretion by stromal cells in all five experiments (Table 1). In Exp 1, the inhibitory effect of IL-1 on PRL production was dose dependent, without causing a significant change in the cell numbers up to a concentration of 2 U/mL (Fig. 1). There was also no difference in cell numbers between stromal cells cultured with and without IL-1 after 12-16 days of culture in the other four experiments (data not shown). In Exp 1, [3H]thymidine uptake of stromal cells cultured in the absence or presence of IL-1 also showed no significant differences (Table 2). Morphological studies showed that spindle-shaped stromal cells (at the start of culture; Fig. 2A) were transformed in the presence of P into large polygonal cells, resembling decidual cells in vivo (Fig. 2B). In contrast, stromal cells cultured with P plus IL-1 remained fibroblastic in appearance even at the completion of culture (Fig. 2C).
Discussion Decidualization is known to be under the control of ovarian steroid hormones. In addition, it has become apparent in various mammalian species that some other factors act as local mediators in the regulation of decidualization, including PGs (1), leukotrienes (2), and platelet-activating factor (16). In humans, however, the existence of local mediators modulating this process has not previously been proven. The present study suggests that IL-1, one of the key mediators of immunological reaction predominantly released from activated macrophages, can work as a local modulator in the decidualization of human endometrium. It has been reported that PRL is produced by the late secretary endometrium of normally cycling women, and that its production correlates with the degree of histological decidualization (17). It has also been reported that PRL is not detected in ESC by immunohistochemistry until they are decidualized (18). Morphologically, decidualization is characterized by the conversion of spindleshaped stromal cells into epithelial-like cells with enlarged nuclei and an increased amount of cytoplasm (19). ESC cultured in the presence of P produced PRL and were transformed into large epithelial-like cells similar to those of in vivo decidua. Irwin et al. (12) have reported that ESC cultured under the influence of ovarian steroid hormones show the ultrastructural and biochemical changes characteristic of decidualization in vivo. These biochemical changes are the production of laminin, fibronectin, and PRL (12). These similarities between Pinduced in vitro changes and in vivo decidualization suggest that this culture system is useful as an in vitro model for decidualization, and that PRL production and the morphological changes detectable in this culture
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 12 November 2015. at 22:37 For personal use only. No other uses without permission. . All rights reserved.
KARIYA ET AL.
1172
JCE & M • 1991 Vol 73 • No 6
TABLE 1. Inhibitory effects of IL-1 on P-dependent PRL production by ESC PRL production (ng/day-106 cells)
Reagentsi P (nmol/L)
IL-1 (U/mL)
Expl, proliferative
Exp2, proliferative
Exp3, proliferative
100 100 100 100 100 100
0 0.002 0.02 0.2 2 20
42.2 ± 2.20 36.1 ± 0.15° 32.6 ± 0.986 26.2 ± 2.18* 11.3 ± 1.15* 11.2 ± 0.856
49.8 ± 1.79 ND ND ND 1.5 ± 0.25* 1.7 ± 0.286
8.6 ± 0.26 ND ND ND 2.9 ± 0.11" ND
Exp4, secretory 31.4 ± 5.38 ND ND ND
