Proc. Natl. Acad. Sci. USA Vol. 90, pp. 11905-11909, December 1993 Physiology Interleukin 6 inhibits mouse placental lactogen II but not mouse placental lactogen I secretion in vitro (trophoblast//cytokine) M. YAMAGUCHI*t, L. OGREN*, J. N. SOUTHARD*, H. KURACHI*, A. MIYAKEt, AND F. TALAMANTES*§ *Department of Biology, University of California, Santa Cruz, CA 95064; and tDepartment of Obstetrics and Gynecology, Osaka University Medical School, Osaka, Japan 565 Communicated by George E. Seidel, Jr., September 7, 1993 (receivedfor review June 9, 1993)

ABSTRACT The mouse produces several poly- members ofthe PRL-GH gene family. We have used primary belonging to the -growth gene fam- cultures of placental cells from several days of pregnancy to ily, including mouse placental lactogen (mPL) I and mPL-II. demonstrate that IL-6 regulates the secretion of mPL-II, but The present study was undertaken to determine whether the not mPL-I, and that the sensitivity of mPL-II secretion to secretion of mPL-I and mPL-H is regulated by interleukin 6 IL-6 varies during gestation. (IL-6), which is present in the placenta and has previously been reported to stimulate the secretion ofpituitary members of this gene family. Effects of human and mouse IL-6 on mPL-I and MATERIALS AND METHODS mPL-II secretion were examined in primary cultures of pla- , Cytokines, and Antisera. mPL-II and recombi- cental cells from days 7, 9, and 12 of pregnancy. IL-6 caused nant mPL-I were purified as described (10, 16). Rabbit a dose-dependent reduction in the mPL-HI concentration in the anti-mPL-I and rabbit anti-mPL-II antisera have been de- medium of cells from days 9 and 12 of pregnancy but did not scribed (11, 16). Recombinant human IL-6 (hIL-6) was a gift affect the mPL-II concentration in the medium ofcells from day from Toshio Hirano and Tadamitsu Kishimoto (Osaka Uni- 7 of pregnancy or the mPL-I concentration in the medium of versity). Recombinant mouse IL-6 (mIL-6) and goat anti- cells from days 7 or 9 of pregnancy. The lowest concentration mIL-6 antiserum were purchased from R & D Systems. of human IL-6 that significantly inhibited mPL-II secretion Cell Dissociation and Culture. Conceptuses were collected was 250 pM. The effect of IL-6 on the mPL-II concentration in from timed-pregnant Swiss Webster mice (Simonsen Labo- the medium was due primarily to inhibition of mPL-II synthe- ratories, Gilroy, CA) on days 7, 9, and 12 of pregnancy sis, which resulted at least partly from a decrease in the (vaginal plug on day 0). The fetus and decidua basalis were steady-state level of mPL-II mRNA. These data raise the discarded from conceptuses from days 7 and 9 of pregnancy, possibility that IL-6 may regulate mPL-I production after and the remaining tissue was dispersed. The labyrinth and midpregnancy in vivo. basal zone were used from conceptuses from day 12 of pregnancy. Cell preparations contained tissue from 130 con- The role of interleukin 6 (IL-6) in regulating the immune ceptuses from day 7 of pregnancy or 65 conceptuses from response and hematopoiesis has been investigated exten- days 9 or 12 of pregnancy. The tissue was dispersed in sively and is well established (1). Recent studies have sug- collagenase, and the cells were fractionated on a Percoll gested that IL-6 is also involved in regulating the function of gradient as described (23). Cells banding at a density of 1.044 several endocrine organs, including the pituitary gland (2, 3) g/ml were suspended in culture medium [NCTC-135 supple- and placenta (4-6). In the rat pituitary gland, IL-6 stimulates the secretion offollicle-stimulating hormone, luteinizing hor- mented with 10 mM Hepes, 25 mM NaHCO3, 1.65 mM mone, prolactin (PRL), and (GH) (2, 3). In cysteine, streptomycin (50 ,ug/ml), and penicillin G (50 the human placenta, it stimulates the secretion of human units/ml)] containing 5% (vol/vol) fetal bovine serum. This chorionic gonadotropin (4-6), which is structurally and func- cell population has been shown (24, 25) to contain trophoblast tionally very similar to luteinizing hormone. giant cells that produce mPL-I and mPL-II. The cells were The of numerous species produce proteins that plated at a density of 2.0-3.0 x 105 cells per cm2 in plastic are members ofthe PRL-GH gene family (for review, see ref. multiwell plates and incubated at 37°C in 95% air/5% CO2. 7). In the mouse, two ofthe placental members ofthis family Two hours after plating, the medium was replaced with are mouse placental lactogen (mPL) I and mPL-II. mPL-I is culture medium without fetal bovine serum. IL-6 was added a 29- to 42-kDa glycoprotein that is present at high concen- to the cultures at this time. In experiments in which IL-6 was tration in the maternal blood between days 8 and 11 of neutralized by pretreatment with anti-IL-6 antibodies, 250 pregnancy (8, 9). mPL-II is a nonglycosylated 22-kDa poly- pM mIL-6 was incubated with goat anti-mIL-6 IgG at 20 that appears in the maternal blood on day 9 of pug/ml for 1 hr at 37°C immediately before addition to the pregnancy. Its concentration increases for several days and cultures. Control cultures in the neutralization experiment then levels offin some strains ofmice or continues to increase were treated with the same concentration of IL-6 that had for the remainder ofpregnancy in others (10-13). The known been incubated with nonimmune goat IgG at 20 pg/ml. The functions ofboth mPLs are like those ofPRL. They stimulate medium was changed daily in all experiments, and cultures mammary gland functional differentiation (14-16) and play a were continued for up to 5 days. role in regulating ovarian steroidogenesis (17). Western Blot Analysis. Western blot analysis was carried Since IL-6 was recently identified in mouse blastocysts, out as described (8) with minor modifications. Briefly, sam- placenta, and uterus (18-22), it was of interest to determine whether this cytokine affects the production of placental Abbreviations: GH, growth hormone; IL-6, interleukin 6; mIL-6, mouse IL-6; hIL-6, human IL-6; mPL, mouse placental lactogen; PRL, prolactin; EGF, epidermal growth factor. The publication costs of this article were defrayed in part by page charge tPresent address: Department of Obstetrics and Gynecology, Osaka payment. This article must therefore be hereby marked "advertisement" University Medical School, Osaka, Japan 565. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 11905 Downloaded by guest on September 30, 2021 11906 Physiology: Yamaguchi et al. Proc. Natl. Acad Sci. USA 90 (1993) 140 ples were subjected to electrophoresis on 12% polyacryl- -A amide gels in the presence of SDS under reducing conditions, 120 and the proteins were transferred to a nitrocellulose mem- brane. The membranes were washed, blocked, and then 100 with rabbit anti-mPL-II antiserum (1:200 dilution). incubated 80 Proteins bound to anti-mPL-II antiserum were visualized by incubating the membranes with 1251-labeled anti-rabbit IgG 60 solution (0.5,uCi/ml; 1 Ci = 37 GBq), followed by autorad- iography using Kodak XAR5 x-ray fim. The autoradiographs 6 40 were scanned with a Bio-Rad model 620 video densitometer. 20 Band intensities were determined with ID ANALYST (Version 3.10) software from Bio-Rad. The specificity of the method 0 was established by replacing the primary antiserum with 0 1 2 3 4 5 nonimmune serum. Recombinant mPL-II was used as a Day of Culture positive control. 120 B Northern Blot Analysis. Cells were seeded into 6-well plates at a density of 3 x 106 cells per well. The cells were harvested 100 with a rubber policeman, and total RNA was prepared by the acid guanidinium thiocyanate/phenol/chloroform method 80 (26). All of the RNA from each well was separated on a 1.5% agarose/formaldehyde gel and transferred to a Nytran mem- X 60 brane (Schleicher & Schuell) (27). 32P-labeled mPL-II cDNA (28) and human ribosomal protein L7 cDNA (27) probes were 40 generated with the Multiprime kit (Amersham). Hybridiza- tions were carried out as described (27). Autoradiography 20 and the analysis of the autoradiographs were carried out as described for Western blot analysis. The intensity of the 0 , mPL-II mRNA band in each lane was normalized to that of 0 1 2 3 4 5 the L7 band to control for differences in sample loading and Day of Culture transfer efficiency between lanes. Protein Synthesis. De novo synthesis of trichloroacetic FIG. 1. Time course of mPL-I secretion by cells from days 7(A) acid-precipitable proteins in the cells and medium was as- and 9(B) of pregnancy in the absence (e) and presence (o) of 2.5 nM hIL-6. Cells were plated at 105 cells per well in 96-well plates and sessed by incubating the cells with 50 ,uCi of [3H]leucine cultured for 5 days. The medium was changed daily and assayed for between 118 and 120 hr of culture as described (25). Intra- mPL-I concentration by RIA. Each value represents the mean ± cellular proteins were extracted with 0.5% Triton X-100. SEM of six wells. There was no difference in the mPL-I concentra- Radnoimmunoassays and DNA Assay. mPL-I and mPL-II tion of the medium between hIL-6-treated and control cells on either concentrations were determined as described (9, 11). The day of pregnancy (P > 0.05). Similar results were obtained in one lowest concentration of mPL-H that could be detected by the replicate experiment for each day of pregnancy. assay was 1.25 ng/ml. When the mPL-II concentration of a sample was less than this value, the sample was assigned a hIL-6 resulted in a significant reduction in the mPL-II con- value of 1.25 ng/ml for statistical analysis. DNA content was centration of the medium (Fig. 2 B and C). In cells from day determined by the method of Hinegardner (29) using calf 9 of pregnancy, the inhibition of mPL-II secretion was thymus DNA as the standard. apparent by the third day of incubation, and maximum Statistical Analysis. Data were analyzed for heterogeneity inhibition of mPL-II secretion occurred on day 5, when the of variance by Bartlett's test. When significant heterogeneity mPL-II concentration of the medium of treated cells was of variance was present, the data were logarithmically trans- -40%o lower than that of control cells in each of two trials. formed to achieve homogeneity of variance prior to addi- Significant inhibition ofmPL-II secretion by cells from day 12 tional analysis; nontransformed data are shown in the figures. of pregnancy in the presence of hIL-6 was also apparent by The effect of IL-6 on the time course of mPL-I and mPL-II the third day ofculture, but the magnitude of the hIL-6 effect secretion was assessed by two-way analysis of variance with was somewhat greater in these cells than in cells from day 9 repeated measures design, with cytokine treatment and day of pregnancy, with the mPL-II concentration of the medium of culture as main effects. When F ratios were significant (P of IL-6-treated cells being 65% and 80%o lower than that of < 0.05), differences between treatment groups on each day of untreated cells in two trials. There was a clear dose-response culture were analyzed by an Ftest. Differences between days relationship in the effect of hIL-6 on mPL-II secretion (Fig. ofculture for a given treatment were not analyzed further. All 3). The lowest concentration of hIL-6 that significantly other data were analyzed by one-way analysis of variance for design, followed by Student- reduced the mPL-II concentration of the medium was 250 completely randomized further inhibition at 2.5 nM. Treatment ofcells with Newman-Keul's test when three or more groups were com- pM, with pared. 2.5 nM mIL-6 inhibited mPL-II secretion to the same extent or more effectively than did hIL-6 (Figs. 2C and 3). Western blot analysis of medium from hIL-6-treated and untreated RESULTS cells confirmed that the anti-mPL-II antiserum used for the Treatment ofcells from days 7 or 9 of pregnancy with 2.5 nM mPL-II RIA recognized a single 23-kDa species in the culture hIL-6 did not affect the mPL-I concentration of the medium medium that comigrated with a mPL-II standard (Fig. 4). The during 5 days ofculture (Fig. 1); the effect of hIL-6 on mPL-I presence of a lower concentration of mPL-II in the medium secretion by cells from day 12 of pregnancy was not tested of hIL-6-treated cells than in the medium of untreated cells because these cells do not produce significant amounts of was also confirmed by the Western blot analysis [mPL-II mPL-I (25). hIL-6 also failed to affect mPL-II secretion by band intensity (arbitrary units): control, 2.05 ± 0.17; hIL-6, cells from day 7 of pregnancy (Fig. 2A). In contrast, incu- 0.79 ± 0.10 (mean ± SEM, n = 3 lanes representing three bation of cells from days 9 and 12 of pregnancy with 2.5 nM wells from one experiment; P < 0.05)]. Downloaded by guest on September 30, 2021 Physiology: Yamaguchi et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11907 20 F A 300

250 a 15 k '200 -b

10 H 150 ~~~100 ~~~~~d 5 k 50

0 0 2 3 0 2.5 25 250 2500 2500 0 1 4 5 mIL-6 Day of Culture hIL-6 (pM) FIG. 3. Effect of IL-6 dose on the mPL-II concentration in the 500 F B medium. Cells from day 12 of pregnancy were plated at 105 per well in 96-well plates and cultured for 5 days in the absence or presence 400 V * of hIL-6 or mIL-6 at the concentrations (in pM) indicated. The medium was changed daily and assayed for mPL-II concentration on 300 the fifth day of culture. Each value represents the mean ± SEM of * six wells. Bars with different letters above them differ significantly (P < 0.05). Similar results were obtained in one replicate experiment, 4 200 , - - _ with the exception that there was no difference in the mPL-II concentration in the medium between cells incubated with 2.5 nM 100 _ hIL-6 and 2.5 nM mIL-6. medium, 81%). The effect of hIL-6 on mPL-II synthesis 0 appeared to be relatively specific since there was no differ- 0 1 2 3 4 5 ence in the amount of newly synthesized trichloroacetic Day of Culture acid-precipitable proteins in cell extracts or medium between control and hIL-6-treated cells. hIL-6 also did not affect cell viability assessed by trypan blue exclusion (data not shown) or DNA content [DNA content (,ug per well) ofcells from day 300 12 of pregnancy incubated for 5 days in the presence or absence of 2.5 nM hIL-6: control, 0.47 ± 0.03; hIL-6, 0.48 ± 0 0.05 (mean ± SEM, n = 6; P > 0.05)]. m 200 Northern blot analysis was performed to determine whether hIL-6 inhibited mPL-II gene expression. Hybridiza- i~im tion of total RNA from cultured cells from day 12 of preg- nancy with a mPL-II cDNA probe yielded a single band of 1 kb (Fig. 5), as observed in Northern blots of total RNA isolated from fresh mouse placenta (28). The intensity of the 0 1 2 3 4 5 signal was significantly greater in blots of total RNA from Day of Culture untreated cells than in those from cells incubated in the presence of hIL-6 for 5 days [mPL-II band intensity normal- FIG. 2. Time course ofmPL-II secretion by cells from days 7 (A), ized to L7 band intensity: control, 2.62 ± 0.41; hIL-6, 0.75 ± 9 (B), and 12 (C) of pregnancy in the absence (s) and presence of 2.5 ± n = 3 lanes representing three wells from nM hIL-6 (o) or 2.5 nM mIL-6 (v). Cells were plated at 105 cells per 0.24 (mean SEM, well in 96-well plates and cultured for 5 days. The medium was one experiment; P < 0.05)] (Fig. 5). changed daily and assayed for mPL-II concentration by RIA. Each To confirm that the inhibitory effect of IL-6 on mPL-II value represents the mean ± SEM ofsix wells. *, Control and treated secretion was due to IL-6 and not to a contaminant ofthe IL-6 groups differ significantly on the same day of culture (P < 0.05). Similar results were obtained in one replicate experiment for cells from days 7 and 9 of pregnancy. In a replicate experiment on cells from day 12 of pregnancy, the mPL-II concentration in the medium of cells incubated with mIL-6 was significantly lower (30-35%) than that of cells incubated with hIL-6 on days 4 and 5 of culture. .3.2.0 To determine whether the reduction in the mPL-II con- centration in the medium of cells incubated with h- or mIL-6 reflected an inhibition of mPL-II synthesis, the mPL-II 21-5 concentrations ofboth the cells and medium were determined after incubation of cells from day 12 of pregnancy with and without hIL-6 (Table 1). The mPL-II concentration of both 1 2 3 4 5 6 7 the cell extract and the medium was significantly lower in hIL-6-treated cells than in controls, indicating that hIL-6 FIG. 4. Western blot analysis. Cells from day 12 of pregnancy the cells did were plated at 3 x 106 cells per well in 6-well plates and incubated inhibited mPL-II synthesis. mPL-II release by in the absence (lanes 1-3) or presence (lanes 4-6) of2.5 nM hIL-6 for not appear to be selectively inhibited by hIL-6 treatment, 5 days. Medium (50 Al) from each of three wells on the final day of since the percentage reduction in mPL-II concentration after culture was subjected to Western blot analysis for mPL-II. Lane 7 IL-6 treatment was similar in the medium and cell extracts contains the purified mPL standard. Positions of molecular mass (percentage reduction in mPL-II concentration: cells, 79%; markers are shown on the right in kDa. Downloaded by guest on September 30, 2021 11908 Physiology: Yamaguchi et al. Proc. Natl. Acad. Sci. USA 90 (1993) Table 1. Effect of hIL-6 on the synthesis of mPL-II and Table 2. Effect of anti-mIL-6 IgG on the inhibition of mPL-II trichloroacetic acid-precipitable proteins secretion induced by mIL-6 Treatment Treatment mPL-II, ng/ml Endpoint Source Control hIL-6 Nonimmune goat IgG 305.4 ± 13.4 mPL-II concentration, Cells 21.0 ± 1.9 4.4 ± 0.1* Nonimmune goat IgG + mIL-6 84.7 ± 6.3* ng per well Medium 108.6 ± 5.8 20.6 ± 0.7* Goat anti-mIL-6 IgG + mIL-6 329.1 ± 28.4 Total 129.5 ± 7.5 24.9 ± 0.7* Cells from day 12 of pregnancy were incubated for 5 days in TCA-precipitable Cells 1.77 ± 0.15 1.90 ± 0.14 medium containing nonimmune goat IgG (20 Ag/ml), nonimmune proteins, cpm Medium 0.42 ± 0.02 0.42 ± 0.03 goat IgG (20 pg/ml) that had been preincubated with 250 pM mIL-6, (xlO-5) per well Total 2.20 ± 0.14 2.32 ± 0.14 or goat anti-mIL-6 IgG (20 pg/ml) that had been preincubated with 250 pM mIL-6. The medium was changed daily. The mPL-II con- Cells from day 12 ofpregnancy were plated into 24-well plates and centration of the medium was determined on the fifth day of culture incubated in the presence or absence of 2.5 nM hIL-6 for 5 days. For by RIA. Each value represents the mean ± SEM ofsix wells. Similar determination of de novo synthesis of total trichloroacetic acid results were obtained in one replicate experiment. (TCA)-precipitable proteins, [3H]leucine was added to the cultures at *Significantly different from each ofthe other treatments (P < 0.05). 118 hr, the cultures were terminated at 120 hr, and the cells and tissue were processed. The mPL-II concentration of the cells and medium differences in the types of cells present in the cultures. At was determined on the fifth day of culture by RIA. Each value midpregnancy the chorioallantoic placenta differentiates, represents the mean ± SEM of three wells. and as pregnancy advances, contributes increasingly to the *Significantly different from the control group (P < 0.05). total cell population ofthe cultures. Thus it is likely that cells preparations, 250 pM mIL-6 was preincubated with anti- from the chorioallantoic placenta are responsive to IL-6 with mIL-6 antiserum prior to its addition to the cells. This respect to mPL-II secretion, whereas placental cells from treatment completely eliminated the ability of mIL-6 to early pregnancy are not. The specific cell types that are inhibit mPL-II secretion (Table 2), indicating that the active responsive to IL-6 are not known. IL-6 may act directly on component of the mIL-6 preparation was mIL-6. cells that already produce mPL-II or are destined to produce the hormone, or the activity of IL-6 could be mediated by factors produced by cells that never express mPL-II. We DISCUSSION have observed a difference in the size of mPL-II-producing These data demonstrate that IL-6 inhibits the secretion of cells between cultures from days 7 and 12 ofpregnancy (ref. mPL-II by placental cells from days 9 or 12 of pregnancy but 25; M.Y., unpublished data). The mPL-II-producing cells in does not affect the secretion of mPL-II by cells from day 7 of cultures of day 7 placenta are larger than those in cultures of pregnancy or the secretion of mPL-I by cells from days 7 or day 12 placenta, as assessed in the reverse hemolytic plaque 9. The effect of IL-6 on mPL-II secretion appeared to be assay and after immunohistochemical staining. It is likely that mediated largely at the level ofhormone synthesis rather than the smaller cells originate from the labyrinth zone, since release since the mPL-II concentration of both the cells and cultures of day 12 placenta would be expected to contain medium was reduced by a similar percentage by IL-6 treat- relatively more cells from this region than would cultures ment. Northern blot analysis suggested that the inhibition of from earlier days of pregnancy (31), and mPL-II has been mPL-II synthesis was due to a reduction in the steady-state localized by in situ hybridization (32, 33) and immunohisto- level of mPL-II mRNA. Since the Northern blot data reflect chemical staining (ref. 34; M.Y., unpublished data) to this total mPL-II mRNA levels in groups of cells from a hetero- region. Since the morphology of mPL-II-producing cells in geneous population, it is not clear whether the amount of vitro differs somewhat between cultures from days 7 and 12 mRNA in individual cells was reduced by IL-6 treatment or of pregnancy, it is tempting to speculate that their respon- whether IL-6 treatment reduced the number of cells express- siveness to PL-II secretagogues may also differ. ing the mPL-II gene. The fact that significant inhibition of IL-6 mRNA and bioactivity are present in the mouse uterus mPL-II secretion did not occur until the third day of culture throughout pregnancy (19-21). IL-6 is produced by uterine suggests that ongoing differentiation of mPL-II cells may epithelial cells, stromal fibroblasts, and leukocytes (22). IL-6 have been inhibited by IL-6. and epidermal bioactivity is also present in the placenta, but IL-6 mRNA has growth factor (EGF) have been reported to inhibit PL-II not been detected in this tissue, suggesting placental IL-6 secretion in rats and mice by inhibiting differentiation of may originate in the uterus (19). The degree to which uterine/ PL-II-secreting cells (23, 30), suggesting differentiation of placental IL-6 regulates the gestational mPL-II profile ofthe these cells could be sensitive to other regulators as well. maternal blood is unknown and will be difficult to determine The increase in the sensitivity of mPL-II secretion to IL-6 until more information is available about the gestational between days 7 and 12 of pregnancy is probably related to profile of placental IL-6 concentrations. To date, placental IL-6 concentrations have been reported only as a function of placental protein content (19), which also changes during the mPL-11 course of gestation. There is a shift at midpregnancy from PL-I to PL-II production in the placenta of both mice and rats (9, 11, 35). -i- ^ In mice this shift results at least partly from a change in gene expression in one population of cells (36). We have shown (23) that EGF may play some role in this shift. EGF stimu- 1 2 3 4 5 6 lates mPL-I production and inhibits mPL-II production in vitro, and the sensitivity of mPL-II production to EGF FIG. 5. Northern blot analysis. Cells from day 12 of pregnancy as were plated at 3 x 106 cells per well in 6-well plates and incubated declines pregnancy advances. The absence of an effect of without (lanes 1-3) or with (lanes 4-6) 2.5 nM hIL-6 for 5 days. Five IL-6 on mPL-I secretion and the increase in sensitivity of to 10 pg of total RNA from each well was subjected to Northern blot mPL-IIsecretion to IL-6 between days 9 and 12 ofpregnancy analysis. The upper row shows a blot hybridized with a 32P-labeled in the present study suggest that IL-6 is probably not involved mPL-II probe, and the lower row shows the same blot rehybridized in regulating this change in PL gene expression. Thus these with a 32P-labeled L7 probe. observations suggest that the relative importance ofEGF and Downloaded by guest on September 30, 2021 Physiology: Yamaguchi et al. Proc. Natl. Acad. Sci. USA 90 (1993) 11909 IL-6 as regulators of mPL-II secretion changes during the 15. Thordarson, G., Ogren, L., Day, J. R., Bowens, K., Fielder, P. course ofgestation. EGF is important in early pregnancy, and & Talamantes, F. (1989) Biol. Reprod. 40, 517-524. IL-6 is important after midpregnancy. 16. Colosi, P., Ogren, L., Southard, J. N., Thordarson, G., Linzer, IL-6 has been reported to stimulate the secretion ofhuman D. I. H. & Talamantes, F. (1988) Endocrinology 123, 2662- chorionic gonadotropin by placental ceils from the first 2667. trimester of pregnancy in the human (4-6), but to our 17. MacLeod, K. R., Smith, W. C., Ogren, L. & Talamantes, F. knowledge, its effects on the (1989) Endocrinology 125, 2258-2266. secretion of PL and other 18. Murray, R., Lee, F. & Chiu, C.-P. (1990) Mol. Cell. Biol. 10, placental members ofthe PRL-GH gene family have not been 4953-4956. assessed previously in humans or in other species. The fact 19. De, M., Sanford, T. H. & Wood, G. W. (1992) Endocrinology that IL-6 inhibits the secretion of mPL-II by the mouse 131, 14-20. placenta suggests that the role of this and other cytokines in 20. Sanford, T. R., De, M. & Wood, G. W. (1992) J. Reprod. regulating PL secretion in other species should be examined. Fertil. 94, 213-220. In humans, IL-6 is produced by normal trophoblast and 21. De, M., Sanford, T. R. & Wood, G. W. (1993) J. Reprod. placental macrophages (37-40), and its production by the Fertil. 97, 83-89. placenta increases as a result of intrauterine infection (41). 22. Robertson, S. A., Mayrhofer, G. & Seamark, R. F. (1992)Biol. These findings raise the possibility that if PL production by Reprod. 46, 1069-1079. the human placenta is inhibited by IL-6, PL secretion could 23. Yamaguchi, M., Ogren, L., Endo, H., Thordarson, G., Kens- be compromised in cases of chronic intrauterine inger, R. & Talamantes, F. (1992) Proc. Natl. Acad. Sci. USA infection. 89, 11396-11400. We thank Drs. M. J. Soares and G. Thordarson for helpful 24. Thordarson, G., Folger, P. & Talamantes, F. (1987) Placenta 8, discussion and Drs. T. Hirano and T. Kishimoto for generously 573-585. providing the human recombinant IL-6 used in these experiments. 25. Yamaguchi, M., Endo, H., Thordarson, G., Ogren, L. & This work was supported by National Institutes of Health Grants Talamantes, F. (1992) Endocrinology 130, 2897-2905. 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