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/pregnancy/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 placenta produces several poly- members ofthe PRL-GH gene family. We have used primary peptides belonging to the prolactin-growth hormone 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- Hormones, 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 growth hormone (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 placentas 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 peptide 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).