Interleukin-1P Regulates Human Cytotrophoblast Metalloproteinase ...

THE

Vol. 269, No. 25, Issue of June 24, pp. 17125-17131, 1994 Printed in U.S.A.

JOURNAL OF B r o w c r c ~ CHEMISTRY ~

Interleukin-1P Regulates Human Cytotrophoblast Metalloproteinase Activity and Invasion in Vitro* (Received for publication, November 2, 1993, and in revised form, March 18, 1994)

Clifford L. LibrachS, Seth L. Feigenbaumg, Kathryn E. Bassn, Tian-yi Cuin, Nicole VerastasP, Yoel SadovskyP, James P. Quigleyll, Deborah L. Frenchll, and SusanJ. Fisher§n**$$ PO From the $Departments of Obstetrics and Gynecology and Reproductive Biology, University of Toronto, Toronto General Hospital, Toronto, Ontario, Canada M5G 2C4, the IDepartment of Pathology, State University of New York at Stony Brook, Stony Brook, New York, and the Departments of §Obstetrics, Gynecology, and Reproductive Sciences, llStomatology, **Pharmaceutical Chemistry, and $$Anatomy, University of California, Sun Francisco, California 94143

access to the maternal circulation by invading the uterinearterial system(2). The depth of invasion is precisely controlled, and errors can have extremely adverse consequences. For example, shallow invasion is associated with preeclampsia (3) and about half thepregnancies that arecomplicated by intrauterine growth retardation (4). Overly aggressiveinvasion, through thedeeper portions of the myometrium and beyond, is associated with placental site tumors, choriocarcinoma, and placenta accreta. Experimental evidence suggests thatnormal trophoblast invasion requires temporal and spatial regulation of the cells’ degradative activity as well as their adhesive interactions. Human cytotrophoblasts produce bothmetalloproteinases,primarily the 92-kDa type IV collagenase (51, and urokinase-type plasminogen activator (6) in vitro. However, only MMP-9* is rate-limiting in tissue culture models of the invasive process (7). The cells’ modulation of specific adhesion molecules involved in cell-cell (e.g. cadherins) and cell-substrate(e.g. integrins) interactions is modulated during invasion. Invasive cyof the calciumtotrophoblastsexhibitdecreasedexpression dependent cell-cell adhesion molecule, E-cadherin (51, and dramatically switch their repertoire of integrin cell-extracellular matrix receptors (2,8). In preeclampsia, where cytotrophoblast invasion of the uterus is abnormally shallow (91, only a subset of cytotrophoblast integrins exhibit the switching pattern observed in normal pregnancy (3), suggesting that the changes normally observed are relatedt o the invasion process. The resultsof these studiesoffer interesting insights into the molecular basis of trophoblast invasion and suggest that factors that regulate92-kDa type IV collagenase expression could have importanteffects on the invasion process. The 5‘-flanking region of the gene for MMP-9 has two putative 12-0-tetradecanoyl-phorbol-13-acetate-responsive elements that could serve as binding sites for the transcription factor AP-1and a consenSuccessful human placentation requires that fetal cells (tro- sus sequence of a transforming growth factor 0 inhibitory elephoblasts) rapidly invade genetically dissimilar maternal tis- ment (10). Recent deletion andmutationanalysis demonsues during the early stagesof pregnancy. To accomplish this strated that three motifs homologous to the binding sites for unusual event, the fetal cells adhere transiently to the uterine the “-1, NF-KB, and Sp-1 proteins contribute positively to epithelium, then aggressively penetrate the decidua and first 92-kDa type IV collagenase induction by 12-0-tetradecanoylthird of the myometrium (1).During thisprocess, they also gain phorbol-13-acetate and tumor necrosis factor a (11). Cell culture systems havebeen used to test the effects of these candi* This work was supported by National Institutes of Health Grants date regulatory molecules. 12-O-Tetradecanoyl-phorbol-13HD 26732 and CA5585201, by the Genesis Research Foundation, by a acetate treatment significantly up-regulated MMP-9 Fellowship from the Medical Research Council of Canada, and by Conproduction by SV-40-transformed fetal lung fibroblasts, the tract DE-AC03-76-SF01012 from the offce of Health and Environmental Research, U. S. Department of Energy. The costs of publication of monocytic leukemia strain U-937 (121, and HT 1080 cells (10, this article were defrayed inpart by the payment of page charges. This 12). Substances that up-regulate 92-kDa type IV collagenase article must thereforebe hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact. $5 To whom correspondence should be addressed: HSW 604, UniverThe abbreviations used are: MMP, matrix metalloproteinase;LPS, sity of California, San Francisco, CA 94143-0512. Tel.: 415-476-5297; lipopolysaccharide; (3-[(3-cholamidopropyl)-dimethylammoCHAPS, Fax: 415-476-4204. niol-1-propane-sulfonate); PAGE, polyacrylamide gel electrophoresis.

During early human pregnancy,fetal cytotrophoblasts rapidly invade the uterus. This process has many similarities to tumor invasion, exceptthat theextent and the timing of cytotrophoblast invasion are carefully regulated. Therefore, this system is particularly useful for studying mechanisms that regulate invasive processes. Previously, we showed that production and activation of the 92-kDa type IV collagenase (matrix metalloproteinase(”P)-9) is necessary for cytotrophoblast invasion in vitro. In other systems, interleukin (IL)-lPis an important regulator of matrix-degrading metalloproteinases. Therefore,we investigated trophoblast production of IL-1P and its receptors, as well as the effects of this cytokine on cytotrophoblast metalloproteinase activity and invasion. The results showed that release of IL-lP parallels the invasive potential of the cytotrophoblasts; the highest levels are produced by first trimester cells and the lowest levels by term cells. Immunoprecipitation showed that cytotrophoblasts express the 80-kDa type I IL-1 receptor, suggesting that autocrine effects are possible. IL-lP stimulated trophoblast MMP-9 secretion (by a mechanism that required nascent mRNA and protein synthesis) as well as metalloproteinase activity and invasion of Matrigel. Increasing (by lipopolysaccharide treatment) or decreasing (by glucocorticoid treatment) IL-lP production had parallel effects on MMP-9 secretion, metalloproteinase activity, and invasion. Because IL-1P and corticosteroids are present in high concentrations at the maternal-fetal interface, normal trophoblast invasion may be regulated, in part, by their opposing actions.In contrast, stimulation of cytotrophoblast IL-1P secretion by lipopolysaccharide may play a role in thesequela of infected fetal membranes.

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expression often positively influence production of other metalloproteinases. However, divergent mechanisms may be involved. For example, in U-937 cells, LPSstimulates transcription of collagenase but not MMP-9. In contrast, the half-life of MMP-9 RNA, but not collagenase RNA, is increased (13). The inhibitory effects of transforming growth factor-p are much less clear. In fact, this growth factor up-regulates the expression of MMP-9 by human keratinocytes (14) and monocytes (15). Additional regulatory factors have also been identified in cell culture systems. For example, IL-1p stimulates production of this metalloproteinase in a number of cell types, including chondrocytes (16), keratinocytes (17), and synovial fibroblasts (18). In contrast, synthetic glucocorticoids inhibit MMP production, either directly, by affecting transcription (19, 20), or indirectly, by inhibiting IL-1 production (21). Because IL-lP and corticosteroids are present in high concentrations at the maternal-fetal interface, they are candidate molecules forregulating trophoblast production of MMP-9 and, consequently, trophoblast invasion. The results presented here support this hypothesis. We found that human cytotrophoblast IL-lp production is regulated in parallel with the invasive potential of the cells and showed that cytotrophoblasts have an IL-1 receptor (type I), suggesting the possibility of autocrine effects. Both IL-1p and LPS, which stimulates production of IL-lp, substantially increased MMP-9 production, as well as metalloproteinase activity and trophoblast invasion in vitro, whereas glucocorticoids had the opposite effect. These results suggest that normal trophoblast invasion may be regulated, in part, by the opposing actions of IL-10 and glucocorticoids. MATERIALSANDMETHODS Cell Isolation and Culture-Cytotrophoblasts were isolated by published methods from first, second (5, 71, and third trimester(7) human placentas. In all cases, after this initial step, the remaining leukocytes were removed by using an antibody to CD-45, a protein tyrosine phosphatase found on bone marrow-derived cells (22), coupled to magnetic beads (7). Previous work from this laboratory showed that this method, combined with the cytotrophoblastisolation procedure we have devised ( 3 , removes contaminating non-trophoblast cells as shown by the absence of classical HLA class I antigens (23). The cells were cultured in serum-free medium (Dulbecco's modified Eagle's medium H21containing 2% Nutridoma-HU (Boehringer Mannheim), 50 pg/ml gentamicin, and 1%glutamine). Fibroblasts were isolated from first trimesterplacentas as previously described (5) and were used after the fourth and before the fifteenth passage. The JAR, BeWo, and JEG human choriocarcinoma cell lines were grown as previously described (5). Buffalo rat liver cells for immunoprecipitation were obtained from American Type Culture Collection and grown in Ham's F-12 medium containing 10% fetal bovine serum. Peripheral blood leukocytes for immunoprecipitation were isolated from wholehuman blood collected byvenipuncture. The blood was layered over Histopaque-1077 (Sigma) and centrifuged (300 x g, 20 rnin). The leukocyte band was collectedand washed 3 times with serumfree medium. Measurement of Secreted Interleukin-lp-Cells (5 x lo5)were plated in 35-mm wells (Costar, Cambridge, MA). The wells were precoated with a mixture of purified human laminin, human fibronectin, and human type IV collagen, each at a concentration of 5 pg/ml. All matrix glycoproteins wereobtained from CollaborativeResearch Inc., Bedford, MA. Stock solutions (lo-' M dissolved in ethanol) of dexamethasone, cortisol, and progesterone were diluted in serum-free medium to obtain the concentrations used for experiments. LPSfrom Escherichia coli serotype 055:B5 (Sigma) was added to serum-free medium at a concentration of 10 ng/ml. After culture for 24 h, the resultant conditioned media were collectedand centrifuged (300 x g ) to remove debris, and the supernatant was frozen a t -70 "C until assayed. IL-1p was measured in triplicate foreachcondition by using an enzyme-linked immunosorbent assay kit (Cistron Biotechnology, Newark, NJ) with a lower limit of detection of 4 pg/ml. A standardcurve was performed for each assay. The intra-assay coefficient of variation was less than 5% for each condition. Immunoprecipitation-Monoclonal antibody to IL-1p was obtained from Cistron Biotechnology, and a monoclonal antibody to the IL-1

Cytotrophoblast Invasion receptor (type I) was obtained from Genzyme Corp. (Boston, MA). Cytotrophoblasts, fibroblasts, peripheral blood leukocytes, and Buffalo rat liver cells (5 x lo6)were cultured as described above for 12 h incysteineand methionine-deficient serum-free medium containing 0.5 mCi T r a r ~ ~ ~ S - l a(ICN b e l ~Biomedicals Inc., Costa Mesa, CA). Conditioned medium was collected and centrifuged, and a proteinase inhibitor mix (phenylmethylsulfonyl fluoride, 1 mM; EDTA, 1 mM; E-64,1 PM) was added. Labeled cells were extracted in lysis buffer containing 0.15 M NaC1,5 m~ EDTA, 50 mM Tris, CHAPS detergent (0.5%),and proteinase inhibitor mix (pH 7.2) for 30 min at 4 "C, after which the lysate was centrifuged for 10 min. To prevent nonspecific adsorption, Protein G-coated Sepharose CL-4B fast flow beads (50 pl; Pharmacia, Uppsala, Sweden) were washed once in IP buffer (Ca2+-and Me-free phosphate-bufferedsaline (pH 7.2), 0.5% CHAPS, 0.02% NaN,, and the proteinase inhibitor mix) and then incubated with continuous agitation for 30 min at 4 "C with unlabeled conditioned medium or cell lysate. After two washes in IP buffer, the beads were incubated with the antibody for 60 min at 4 "C with continuous agitation and then washed twice in IP buffer, producing antibody-coupled protein G beads. To remove proteins that adsorb nonspecifically, the labeled conditioned media or lysates were mixed with uncoupled protein G beads (100 p1) and incubated for 30 min a t 4 "C with continuous agitation. The precleared samples were then incubated with the antibody-coupledbeads for 1h at 4 "C with continuous agitation. The beads were then washed three times in IP buffer and once in a low salt buffer (20 m~ Tris (pH KO), 0.5% CHAPS, and proteinase inhibitor mix). Before electrophoresis,the beads were boiled in sample buffer for5 min. They werethen subjected t o SDS-PAGE (241, after which the gels were fixed in a solution of 10% glacial acetic acid, 45% methanol, and 0.1% glycerolovernight. Next, the gels weresoaked first in EN3HANCE autoradiography enhancer (Du Pont NEN) for 1h and then incold water for 30 min. They werethen dried and exposed to x-ray film (KodakX-OMAT A R ) . Interleukin-1 Receptor Binding Assay-Cytotrophoblasts (5 x lo5) were plated on 16-mm wellsand cultured in serum-free medium for 12 h before the start of the assay. The wellswerewashedtwice with serum-free medium, and then increasing concentrations of lZ5I-labeled IL-lp were added to each well. Cells were then incubated for 3 h at 37 "C. Duplicate samples were analyzed, and the experiment was repeated on three separate occasions. Following the incubation, 1 ml of phosphate-buffered saline containing 0.5% bovine serum albumin was added to each well; cells were then incubated for 30 min at room temperature. Next, two washes in ice-cold phosphate-buffered saline were performed in succession to remove the remaining free labeled protein. The cells were lysed, and radioactivity was determined in a Packard Cobra gamma counter. Saturation binding analysis was performed by fitting the untransformed data t o a saturation isotherm (rectangular hyperbola) (25),using Igor software (Wavemetrics Corp.,Portland, OR). Substrate Gel Zymography for the Detection of MetalloproteinusesMetalloproteinases in cytotrophoblast extracts and conditioned media were assayed using substrate gel zymography as previously described (7). Stromelysins and interstitial collagenase were best displayed in casein-containing gels, whereas the 72- and the 92-kDa type IV collagenases were best detected in gelatin-containing gels. Western Blotting to Study Regulation of 92-kDa Qpe N Collagenase Production-First trimester human cytotrophoblasts were cultured as described above. Actinomycin D (5 pg/ml; Sigma) or cycloheximide (5 pg/ml; Sigma) were added to both control cultures and cells cultured in the presence of dexamethasone, lipopolysaccharide, or IL-lp. Conditioned medium samples were collected after 24 h in culture, stored at -80 "C, and analyzed by both zymography (as described above)and by immunoblotting. For immunoblotting, conditioned medium(10 pl) was solubilized in loading buffer, subjected to SDS-PAGE (10% gels), and transferred by blotting t o nitrocellulose (26).Nonspecific reactivity was blocked by incubating the transfers for 1 h in Tris-buffered saline containing Tween-20(TBST; 0.01 M Tris-HC1, 0.15 M NaC1, and 0.05% Tween-20, pH8.0) and 5%non-fat dried milk (Carnation). The transfers were then incubated for 2 h at 25 "C with anti-MMP-9antibody (7-11, 27), which was suspended (5 pg/ml) in TBST containing 5% non-fat dried milk. The blots were washed three times (10 min each time) in TBST. Primary antibody was detected by incubating the blot for 2 h at 25 "C with horseradish peroxidase-conjugated rabbit anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:5000in TBST containing 5% non-fat dried milk. The membranes were washed in TBST, rinsed in phosphate-bufferedsaline, and developed for enhanced chemiluminescence with a kit (Amersham Corp.). Measurement of Metalloproteinase Actiuity-Total metalloproteinase activity was measured by using the thiopeptolide Ac-Pro-Leu-Gly-

IL-lPRegulates Human Cytotrophoblast Invasion

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Cell Type FIG.1. IL-lp production by human cytotrophoblasts is developmentally regulated. Among primary human cytotrophoblasts, first trimester (1st T M )cells secreted (per cell) the most IL-lp, and term (3rd 2 " ) cells secreted the least. Barely detectable amounts of this cytokine werefound in theconditioned mediafrom choriocarcinoma cell lines (JEG, JAR, BeWo) and fibroblasts isolated from first trimester human placentas (Plac. FIBS).Bars = S.E.

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FIG.2. First trimester human cytotrophoblasts express the SCH[CH,CH(CH,),lCO-Leeu-Gly-OC~H5 (Bachem, Philadelphia, PA) in type I IL-1 receptor. Human placental fibroblasts (lane I ) and first a spectrophotometric assay describedby Weingarten et al. (28). Invasion Assay-Invasion was quantified in Matrigel (Collaborative trimester cytotrophoblasts (lane 2) were labeled with T r a ~ ~ ~ ~ S - l a b e l ~ " . Researchkcoated Transwell inserts (6.5 mm; Costar) containing poly- Immunoprecipitation of cell extracts was performed with an antibody carbonate filters with 8-pm pores as previously described (7). Briefly, that recognizes the type I IL-1 receptor. The resulting proteins were the substanceto be tested was mixed with the Matrigel and added to the separated by SDS-PAGE and visualized by autoradiography. Both fibroblastandcytotrophoblastimmunoprecipitatescontained a n M, medium bothabove and below the Matrigel-coated filter.First trimester 80,000 band consistent with the molecular weightof the type I receptor. cytotrophoblasts (2x lo5)were platedon the inserts and cultured for 24 The fibroblast immunoprecipitates contained lower (M,30,000) and h. The cells were fixed as previously described, and the filters were higher (M,220,000) molecular weight proteins, which may be related to analyzed without dryingby using a charge-free anticontamination sys- additional IL-1-binding proteins of similar estimated molecular weights tem mounted on a n SX-40Ascanning electronmicroscope (International that have been describedpreviously (31).Buffalo rat liver cells, which ScientificInstruments,Pleasanton, CA). Fiverepresentative photo- fail to express this receptor (lane 3 ) ,were used a s a negative control. graphs (150x) of each filter underside were taken, and the surface area covered by invasive cells was estimated with an overlay grid(29). For each condition, at leasttwo filters were examined per experiment, and ent with the molecular weight of IL-16 (153 amino acids, M, 17,400). No labeled proteins were immunoprecipitated from the experiments were repeatedon at least three separateoccasions.

placental fibroblast-conditioned medium. Cytotrophoblasts Express Vpe Z ZL-lp Receptors-We then Human Cytotrophoblast Production of ZL-lp in Vitro Is Zn- determined whether cytotrophoblasts have the type I IL-1p versely Related to the Gestational Age of the Placenta from receptor and, thus, the potential torespond to the IL-1p they Which the Cells Are Isolated-IL-lp levels in conditioned me- produce. Todo so, we used a monoclonal antibody that recogdium collected from cytotrophoblasts during the first 24 h of nizes the type I receptor to immunoprecipitate this protein culture were quantified by enzyme-linked immunosorbent as- from 35S-labeledcytotrophoblast lysates. Human placental fisay. An important aspect of these experiments was that all broblast and Buffalo rat liver cell lysates were processed in assays used highly purified preparations of cytotrophoblasts parallel as positive and negativecontrols, respectively. The from which bone marrow-derived cells were removed as de- results of SDS-PAGE and autoradiographic detection of the scribed under "Materials and Methods." IL-1p production by labeled proteins areshown in Fig. 2. In agreementwith studies purified cytotrophoblasts in culture depended on the gestashowing that humanfibroblasts express the 80-kDa IL-1p type tional age of the placentas from which the cells were isolated I receptor (30),a 35S-labeledprotein of M,80,000 was immuno(Fig. 1).Cytotrophoblasts isolated from first trimester placen- precipitated from placental fibroblast lysates (lane 1).Howtas produced 2.4 and 3.8 times more IL-lp than thoseisolated ever, lower (M,30,000) and higher (M,220,000) molecular from second and third trimesterplacentas, respectively. In par- weight proteins were also detected that may be related to adallel experiments, we examined IL-1p production by human ditional IL-1-binding proteins of similar estimated molecular choriocarcinoma cell lines (JEG,JAR, and BeWo) and by fibro- weights that have been described previously (31). First triblasts isolated from first trimester human placentas (Fig. 1). mester cytotrophoblast immunoprecipitates (lane 2) contained All four cell lines produced barely detectable amountsof IL-1p a single band of M,80,000, suggesting that thesecells express that were, at the highest level, 4 % of the amount secreted the type I IL-1p receptor. As expected, no proteins were deduring the same time period by first trimester humancytotro- tected inthelysatesprepared from Buffalo rat liver cells phoblasts. Thus, among the placental cells studied, primary (lane 3). cultures of first trimester human cytotrophoblasts produced We also determined the Kd of cytotrophoblast IL-1 receptor the most IL-1p and the various other cell lines produced the binding and the estimated number of receptor siteskell by analeast. lyzing the binding of 1251-labeledIL-lp ligand to intact first To confirm IL-1p production by cytotrophoblasts, we used a n trimester cytotrophoblasts. IL-1p binding to cytotrophoblasts IL-lp-specific antibody to immunoprecipitate 35S-labeled pro- revealed one class of binding affinity with a Kd of 310 PM, teins from the medium in which first trimester humancytotro- comparable to that determinedfor human monocytes (600 PM) phoblasts, human leukocytes (positive control), and placental (32) and human peripheralT-lymphocytes (280 PM) (33). There fibroblasts (negative control) had been cultured for 24 h (data were approximately 6.6 x lo3 binding siteslcell, approximately not shown). Autoradiographic detection of immunoprecipitated 30-fold more sites than in nontransformed cells taken from 35S-labeledproteins from both cytotrophoblast- and leukocyte- tissues and circulating blood (34) and comparable to therecepconditioned media showed a prominent M,17,000 band consist- tor density of transformed cells (35). RESULTS

IL-1p Regulates Human Cytotrophoblast Invasion

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FIG.4. r C l P significantly increases cytotrophoblast MMP-9necretion by a mechanism that requires nascent protein and RNA synthesis. A. as compared with crlls that w r r r incuhatrd in rnrdlurn alone Ilanr 1 ) . medium from cytotrophohlasts t h a t wrrr culturrtl in the presence of I L l p tlnnr 2 I contained approximately twice t h r amount of MMP-9 protein. In addition, lower molecular wright. immunorractivr bands (60-85 kDaI, characteristic of 92-kI)a type I\' collagenasr activation, were more prominent in samples of conditioned mcdlum from the cytokine-treated cultures. H , t r r n t m r n t of t h r cultures with r i t h r r cycloheximide alone tlanr 2 I or both cycloheximide and 11,-1[1 I lnnr 3 I meatly reduced MMP-9 synthesis. Likewise. t r r n t m r n t of t h r cultures with actinomycin D had a similar effect on hoth control culturrq f l n n r 4 J and those treated with I L l D clnnr 5 1.

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rified first trimester human cytotrophohlastx2 Thus, is it likely t h a t t h eglucocorticoid inhihitory effect occursvia a c-ytotrophoblast glucocorticoid receptor. Finally, progesterone,in high con0 -10 - 9 10-7 10-6 10-5 0 10 10 centrations such as those postulated to exist at the maternalCortisolConcentration (M) fetal interface, can competitively inhihit glucocorticoid-receptor interactions(reviewedin Ref. 381. We investigatedwhether high concentrations of this steroid hormone could reverse cortisol inhibition of cytotrophoblast I L l P production. Progesterone added alone (10.'' M I had no significant effect (Fig. X I . p 1000 However, when added with cortisol, progrsterone partially red versed the profound inhihitory effect of corticosteroids on cy800 0, totrophoblast I L l p production (Fig. 3C ). a 600 FirstDimesterCytotrophohlastProduction of thc 92-kDn M T)pe IV CollaRenase Is Stimulated hv II!-ID a n d R q r r i r r sXnn400 cent RNA a n d Protein S~vnthesis-Pre~ously. we drtermined which metalloproteinases c-ytotrophohlasts produce in r4tro by 200 gel (caseinandgelatin1zymography We usingsubstrate 0 showed that these cells synthesize the 92-kDa type IV collaControl PrOg Cortisol Cortisol (10-6) genase almost exclusively ( T i , 7). Furthermore. thp activity of Concentrstlon (M) (10-5) (10-6) Prog (10-5) this metalloproteinase was rate-limiting for human trophoblast FIG.3. Cortisol and LPS affect I L l p production by first trimester human cytotrophob1asts.A.LPS stimulated c-ytotrophohlast invasion in vitro(7). Because IL-lp up-regulates the production production of I L l p by approximately 5-fold. H , treatment of t h e cells of metalloproteinases in other cells ( 16, 171, we examined how with cortisol resulted in a dose-dependent decrease in I L l p production substances thatmodify c-ytotrophohlast I L l P production affect by first trimester human cytotrophohlasb i n t i f r o . Maximum effects cytotrophoblast metalloproteinase synthesis, using substrate were observed at cortisol concentrations of 10.' ~1 and above. C , proges- gel zymography to determine which MMPs were produced.[{.'e terone (Prog) alone had no significant effect.However, progesterone of LPS partially reversed the profound inhibitory effect of corticosteroids on found that when cytotrophohlasts were exposed to levels (10 ng/ml) that had stimulated IL-1P production, the 92-kDa cytotrophoblast I L l p production. Dnrs = S.E. type IV collagenase was again the predominant c-ytotrophohlast FirstnimesterCytotrophoblastProduction of IL-IP Is metalloproteinaseinboth cell extractsandconditionedmeStimulated by LPS and Inhibited by Corticosteroids-Next, we dium; no other members of this proteinase family had heen investigated whether two factors, LPS (36) and corticosteroids induced (data not shown). In contrast, dexamethasone greatly (37), known to be potent regulators of IL-1P production both in reduced MMP-9 production. Because zymography is not rigorvitro and in vivo in other cells, had similar effects on cytotroously quantitative, we used Western hlotting to more precisely phoblast productionof this cytokine. Additionof LPS (10 ng/ml) determine the effects of IL-Is. LPS. and dexamethasone on a 5-fold increasein the cytotrophohlastproduction of MMP-9.Densitometryshowed to the culture medium resulted in amount of IL-lj3 released into the conditioned medium during that compared with control cultures (Fig. 4 4 , Innr I ). cells the first 24 h of culture (Fig.3A ). Conversely, addition of either cultured in the presence of I L l p (Fig. lane 2 1 secreted cortisol (Fig. 3 B ) or dexamethasone (data not shown) resulted approximately twice the amount of the 92-kDaform of MMP-9. in dose-dependent inhibitionof cytotrophoblast IL-1P producInaddition,lowermolecularweight.immunoreactivehands tion. The largest inhibitory effectwas achieved a t cortisol con- (60-85 kDa), characteristic of 92-kDa type I\' collagenase acM andaboveand at dexamethasoneconcencentrations of tivation, were more prominent in samples of conditioned metrations of IO-" M and above. Viability (>95%), determined as dium from the cytokine-treated cultures.A similar increase in the ability of the cells to exclude trypan blueat the end of the MMP-9secretionwasobservedinLPS-containingcultures, incubation period, was no different between the control and the highest glucocorticoid concentrations. We have recently dem' S. L. Feigenbaum, C. L. Lihrach. P. Siiteri. and S..J. Fishrr, unpuhonstrated the presenceof glucocorticoid receptors in highly pu- lished observations. 200

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0 .10”0 lo-’‘ IO-* 10” 10 ng

FIG.6. IL-1/3 and LPS, which stimulates cytotrophoblast production of IL-la, stimulate cytotrophoblast invasion in vitro, whereas dexamethasone treatment has the opposite effect. Bars = S.E.

trigel and to the culture medium prevented cytotrophoblast at lo-’ M. Progesterone alone invasion, with maximal inhibition M) had no significant effect but could partially block inhibition by corticosteroids (data not shown), again suggestingan important role for both the glucocorticoid receptor and this steroid hormone in mediating the observed effects. In contrast, addition of either IL-1p or LPS to the culture medium significantly increased(by approximately 50%)cytotrophoblast invaDexamethasone Concentration (M) sion (Fig. 6). Because scanning electron microscopy was used to quantify FIG.5. LPS stimulates and dexamethasone inhibits cytotrophoblast metalloproteinase activity. A, LPS treatment increased invasion, we examined the morphology of the cells in the presmetalloproteinase activity in experimental conditioned mediumas com- ence of all the substances tested (data shown). not Our previous pared with control ( p < 0.05, Student’s t test). B, in contrast, dexawork showed that during the invasion process, trophoblast methasone inhibited metalloproteinase activity, with maximum effects cells, which normally exhibit an epithelial appearance, have observed at concentrations a t or above M. Burs = S.E. highly elongated shapes and numerouslong, complicated prowhereas those treated with dexamethasoneshowed at least a cesses that interact with the extracellular matrix (7). In the 4-fold reduction in secretion of this metalloproteinase (data not presence of glucocorticoids, the morphology of the cells was significantly altered. They were round, rather than elongated, shown). Next, we examined the mechanismby which IL-1p controls and formed few processes. In contrast, additionof either IL-lp MMP-9 secretion. Addition of cycloheximide to the medium in or LPS, stimulatorsof invasion, resulted in cells with theelonprocesses that are characteristic of which the cytotrophoblasts were cultured nearly abolished 92- gated shapes and numerous kDa typeIV collagenase secretion in bothcontrol cultures (Fig. invasive cytotrophoblasts both invitro and in vivo (3). 4 B , lane 2) and in those containing IL-1p (Fig. 4 B , lane 3). DISCUSSION Similarly, addition of actinomycin D to the cultures greatly reduced MMP-9 production(Fig. 4 B , lanes 4 and 5). These Our results show that human cytotrophoblasts, highly puriresults show that IL-10stimulation of cytotrophoblast MMP-9 fied to remove bone marrow-derived cells, produce IL-1p in secretion in vitro requires nascent RNA and protein synthesis. vitro. Release of this cytokine into the culture medium is deFirst DimesterCytotrophoblast MetalloproteinaseActivity Is velopmentally regulated; the highest levels are produced by Stimulated by LPS and Inhibited by Dexamethasone-Because cytotrophoblasts from first trimester placentas and thelowest synthesis of metalloproteinases does not necessarilymean they levels by cytotrophoblasts isolated from term placentas. In adare converted to their active forms, we used a very sensitive dition, human cytotrophoblasts express the type I IL-1 recepchromogenic assay (28) to quantify metalloproteinase activity. tor, whichmeans thatsome of the effects mediated by the IL-lp The chromogenic assay showed that LPS treatment also sig- they produce may be autocrine. We also found that factors that nificantly increased metalloproteinase activity (Fig. 5 A ) . Con- either enhance (LPS) or inhibit (glucocorticoids) cytotrophoM) (Fig. 5 B ) and cortisol (lo-’ M) blast IL-1p production have a parallel effect on the cells’ proversely, dexamethasone (data not shown) inhibited MMP-9 production and metallopro- duction of MMP-9, as well as on metalloproteinase activity and teinase activity. cytotrophoblast invasion in vitro. These results suggest that Dexamethasone, LPS, a n d Exogenous I L - l p Alter Cytotro- human trophoblast invasion may be controlled,at least in part, phoblast Invasion inVitro-Next, we determined whether fac- by the IL-1p they produce andor encounter at the maternaltors that alter cytotrophoblast metalloproteinase activity can fetal interface. affect an importantbiological property of these cells, the ability Previous studies have used immunolocalization techniques to rapidly invade extracellular matrices onwhich they are to determine which placental cells produce IL-1p in vivo. The plated. To do so, we used an in vitro assay of invasive potential human placenta has three major trophoblast populations, each in which the abilityof cytotrophoblasts to invade extracellular constituting a different stage in the trophoblast differentiation matrix (Matrigel)-coated filtersandemergethrough 8-pm pathway. Cytotrophoblast stem cells, most numerous in early pores onthe filter underside is quantified (7).Addition of either gestation human chorionic villi, can either fuse to form the cortisol (data not shown) or dexamethasone (Fig. 6) to theMa- multinucleate syncytium thatcovers the floatingvilli or aggre-

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gate to form the cell columns of the anchoring villi; the latter give rise to the subpopulation of cytotrophoblasts that invade the uterus(2). Paulesu et al. (39), using immunocytochemistry, showed that themultinucleate syncytium and invasive cytotrophoblasts, but not the villus cytotrophoblast stem cells, contain immunoreactive IL-1p. Furthermore, stainingfor this cytokine was strongest during the first trimester. Hu et al. (40) detected IL-1p in both floating villus cytotrophoblasts and syncytiotrophoblasts, but did not examine its expression by the invasive subpopulation of cells. As to in vitro production of this cytokine, previous reports suggested that isolated term cells secrete IL-1p (41). However, no attempts have been made to determine whether these levels change during gestation. Our finding suggests the possibility that their production of IL-1p in vitro is, at least in part, the result of differentiation of these cells along the invasive pathway. Wenow know that differentiation of early gestation cytotrophoblasts in cultureis skewed in thedirection of invasiveness and awayfrom syncytium formation. For example, cultured first trimester cytotrophoblasts, but not term cells, rapidly switch their integrin cell-matrix adhesion molecules3so that after 2days, they express the repertoire that ischaracteristic of invasive rather thanvillus cytotrophoblasts (2). In contrast, starting in early second trimester, the ability of these cells to differentiate along the invasive pathway in vitro slows dramatically, until at term it islost completely. The hypothesis that IL-lp production is up-regulated during differentiation and invasion is consistent with previous studies that showed immunoreactive IL-1p in invasive cytotrophoblasts (39) and message for this cytokine in the decidua (421, which is populated by invasive cytotrophoblasts. A number of functions have been described for the interleukin-1 family of proteins (IL-la, IL-lp, and IL-1-receptor agonist). IL-1 induces fever, sleep, anorexia, and hypotension but can also stimulate host defense properties (for a recent review, see Ref. 43). The effects of IL-lP on trophoblast function have also been studied, particularly its impact on hormone production. IL-lp stimulates secretion of human chorionic gonadotropin by cytotrophoblasts (44), probably by activating IL-6 and the IL-6 receptor system (45). In addition, it stimulates trophoblast aromatase activity (46), as well as the release of corticotropin-releasing factor and adrenocorticotropin hormone from the placenta (47). Our results suggest that IL-lPcan also affect cytotrophoblast invasion, most likely by regulating activity of the 92-kDa type IV collagenase. Trophoblast expression of IL-lp receptor proteins has not been investigated. Two IL-1 receptors, types I and11,have been described (48). The type I receptor functions in signaling (49). For example, IL-1 induces NF-KBthrough the type I, but not the type 11, receptor in lymphocytes (50). In contrast, the type I1 receptor inhibits IL-1 activity by acting as a decoy target (51).Having found that cultured first trimester humancytotrophoblasts express a relatively large number of type I IL-1p receptors, we then showed that one means by which the cells respond to this cytokine is by increasing MMP-9 production and, consequently, their invasive potential. That the highest levels of IL-1p production occurat the stage when cytotrophoblasts have a large number of IL-1p receptors suggests that the developmental regulation of cytotrophoblast invasion observed both in vitro (7) andin vivo (3) may be, at least in part, under autocrine control. Our finding that substances (e.g. glucocorticoids, LPS) that influence IL-1 production have a profound impact on trophoblast metalloproteinase activity and invasion has interesting implications. The effects of glucocorticoids on placental funcC. H. Damsky and S. J. Fisher, manuscript in preparation.

tion are less well studied than the effects of IL-1. High maternal glucocorticoid levels are correlated with large placentas (52), suggesting that the placenta can respond to maternal glucocorticoids in vivo. Isolated cytotrophoblasts have a functional glucocorticoid receptor (53). Glucocorticoid treatment of these cells stimulates expression of corticotropin-releasinghormone (53) and human chorionic gonadotropin (a and p subunits) (54). Because maternal free cortisol levels increase during pregnancy (55), the negative effects of glucocorticoids on trophoblast metalloproteinase production and invasion may be an example of how maternal (paracrine) factors influence cytotrophoblast differentiation along the invasive pathway. In contrast, LPS may playa role in pathological situations, rather than innormal placental function. For example, a role has been proposed for IL-1p in theinitiation of pre-term labor associated with infection of the amniochorion (56). Cytotrophoblast invasion involves the regulated expression and/or activation not only of proteinases ( 5 , 7) but also of adhesion molecules(2, 3, 5 ) and the cytotrophoblast-specific class I molecule, HLA-G (23, 57). It is possible that IL-1p and substances that regulate its production affect trophoblast expression of adhesion molecules as well as their MMP-9 activity and invasion. For example, IL-1p regulates integrin expression in other cell types, including human endothelial cells (58). Finally, placental bed immune interactions are probably also extremely important in controlling cytotrophoblast invasion. It is likely that IL-1p is one of many different cytokines that participate in the cross-talk between maternal and fetal cells (reviewed in Ref. 59). In summary, we report the results of experiments in which we have begun t o study the effect of substances that may regulate cytotrophoblast metalloproteinase production and, thus, could playa hierarchical role in controlling human cytotrophoblast invasion. As in some pathologicalsituations (e.g. rheumatoid arthritis) where cytokine-stimulated collagenase activity is modulated by glucocorticoids,we found that these two regulatory mechanisms have opposing effects on trophoblast metalloproteinase activity. It is tempting to speculate that when cytotrophoblasts are rapidly invading the uterus during early pregnancy, the balance is tipped in the direction of mechanisms that stimulate invasion, including stimulation of trophoblast metalloproteinase activity by IL-1p. Therefore, we would predict that when invasion stops later ingestation, the balance is tipped in the otherdirection by inhibitory mechanisms, which include the action of maternal glucocorticoids. Acknowledgment-We thank Dr. Penti Siiteri for helpful discussions and gifts of steroid hormones.

REFERENCES 1. Pijnenborg, R. (1990) lboph. Res. 4,33-50 2. Damsky, C. H., Fitzgerald, M. L., and Fisher, S. J. (1992) J. Clin. Invest. 89, 210-222 3. Zhou, Y.,Damsky, C. H., Chiu, K., Roberts, J. M., and Fisher, S. J. (1993) J. Clin. Inuest. 91, 950-960 4. Gerretsen, G.. Huisjes, H. J., and Elema, J. D. (1981) J . Obstet. Gynaecol.Br. Commonw. 88,876-881 5. Fisher, S.J., Cui, T-Y., Zhang,L., Hartman, L., Grahl, K., Zhang, G-Y., Tarpey, J., and Damsky, C. H. (1989) J. Cell Biol. 109,891-902 6. Queenan. J. T.. Jr.. Kao. L.-C..Arboleda. C. E.. Ulloa-Amine,A., Golos, T.G., Cines,’D. B.;and Straws, J. F., 111 (1987) B i d . Chem. 262,10903-10906 7. Librach,C.,Werb, Z., Fitzgerald, M. L., Chiu, K., Comin, N., Esteves, R., Galardy, R., Damsky,C. H., andFisher, S. J. (1991) J. Ce22 B i d . 113, 437449 8. Aplin, J. D. (1991) J. Cell Sci. 99, 681-692 9. Redman, C. W. G. (1991) Placenta 12,301408 10. Huhtala, P.,Tuuttila,A,, Chow, L. T., Lohi, J., Keski-Oja, J., and Tryggvason, K. (1991) J. B i d . Chem. 266, 16485-16490 11. Sato, H., and Seiki, M. (1993) Oncogene 8,395-405 12. Wilhelm, S. M.,Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A., and Goldberg, G. I. (1989) J. Biol. Chem. 264, 17213-17221 13. Saarialho-Kere.U. K., Welgus, - . H. G., and Parks, W. C. (1993) J. Biol. Chem. 268,17354-17361 14. Salo, T.,Lyons, J. G., Rahemtulla, F., Birkedal-Hansen,H., and Lajava, H.

IL-1p Regulates Human Cytotrophoblast Invasion (1991) J. Biol. Chem. 266, 11436-11441 15. Wahl, S . M., Allen, J. B., Weeks, B. S., Wong, H. L., and Klotman, P. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,4577-4581 16. Ogata, Y., Pratta, M. A,, Nagase, H., a n d h e r , E. C. (1992)Exp. Cell Res. 201, 245-249 17. Lyons, J. G., Birkedal-Hansen, B., Pierson, M. C., Whitelock, J. M., and Birkedal-Hansen, H. (1993) J. Eiol. Chem. 268, 19143-19151 18. Unemori, E. N., Hibbs, M. S., and Amento, E. P. (1991) J. Clin. Inuest. 88, 1656-1662 19. Werb, Z. (1978) J. Exp. Med. 147, 16961712 20. Frisch, S.M., and Ruley, H. E. (1987)J . Biol. Chem. 262, 16300-16304 21. Butler, L. D., Layman, N. K., Riedl, P. E., Cain, R. L., Shellhaas, J., Evans,G. F., and Zuckerman, S. H. (1989) J. Neuroimmunol. 24, 143-53 22. Charbonneau, H., Tonks, N. K., Kumar, S., Diltz, C. D., Harrylock, M., Cool, D. E., Krebs, E. G., Fischer, E. H., and Walsh. K. A. (1989) Proc. Natl. Acad. Sci. U. S. A . 86, 5252-5256 23. Kovats, S., Main, E. K., Librach, C., Stubblebine, M., Fisher, S. J., and DeMars. R. (1990) Science 248. 220-223 24. Laemmli, U. K. (1970)Nature 277,680-685 25. Murlas, C., Nadel, J. A,, and Roberts, J. M. (1982) J. Appl. Physiol. 52,10841091 26. Towbin, H., Staehelin, T., and Gordon, J . (1979)Proc. Natl. Acad. Sci. U. S. A. 76,4350-4354 27. Ramos-DeSimone, N., Moll, U. M., Quigley, J . P., and French, D. L. (1993) Hybridoma 12, 349-363 28. Weingarten, H., Martin, R., and Feder, J. (1985) Biochemistry 24, 67304736 29. Weibel, E., and Bolender, R. (1973) in Principles and Techniques for Electron Microscopy (Hayat, M. A., ed) Vol. 3, pp. 237-261, Van Nostrand Reinhold Co. Inc., New York 30. Chin, J., Cameron, P. M., Rupp, E. A,, and Schmidt, J. A. (1987) J. Exp. Med. 165,7046 31. Dinarello, C. A. (1991) Blood 77, 1627-1652 32. Uhl,J.,Newton, R. C., Gin, J. G., Sandlin, G., and Horuk, R. (1989) J. Immunol. 142,1576-1581 33. Shirakawa, F., Tanaka, Y., Ota, T., Suzuki, H., Eto, S., and Yamashita, U. (1987) J . Immunol. 138,4243-4248 34. Horuk, R., Huang, J. J., Covington, M., and Newton, R. C. (1987) J . Biol. Chem. 262, 16275-16278 35. Savage, N., Puren, A. J., Orencole, S. F., Ikejima, T., Clark, B. D., and Dinarello, C. A. (1989) Cytokine 1, 23-35 36. Newton, R. C. (1986) J. Leukocyte Biol. 39, 299-311 ~~

17131

37. Knudsen, B. J., Dinarello, C. A., and Strom, T. B. (1987) J . Immunol. 139, 41294134 38. Spitz, I. M., and Bardin, C. W. (1993)N. Engl. J. Med. 329, 404-412 39. Paulesu, L., King, A,, Loke, Y.W., Cintorino, M., Bellizzi, E., and Boraschi, D. (1991) Lymphokine Cytokine Res. 10,443448 40. Hu, X-L., Yang, Y., and Hunt, J. S. (1992) J. Reprod. Immunol. 22, 257-268 41. Main, E., Strizki, J., and Schochest., P. (1987) Doph. Res. 2, 149-160 42. Kauma, S., Matt, D., Strom, S., Eierman, D., and Turner, T. (1990)Am. J . Obstet. Gynecol. 163, 1430-1437 43. Dinarello, C. A,, and Wolff, S. M. (1993) N . Engl. J. Med. 328, 106-113 44. Yagel, S., Lala, P. K , Powell, W. A,, and Casper, R. F. (1989)J. Clin. Endocrinol. & Metab. 68, 992-995 45. Masuhiro, K., Matsuzaki, N., Nishino, E., Taniguchi, T., Kameda, T., Li, Y., Saji, F., and Tanizawa, 0.(1991)J . Clin. Endocrinol. & Metab. 72,594401 46. Nestler, J. (1993) Endocrinology 132,566-570 47. Petraglia, F., Garuti, G. C., DeRamundo, B.,Angioni, S., Genazzani, A. R., and Bilezikjian, L. M. (1990)Am. J. Obstet. Gynecol. 163, 1307-1312 48. Dinarello, C. A,, Clark, B. D., Puren, A. J., Savage, N., andRosoff, P. M. (1989) Immunol. Today 10,49-51 49. Dower, S. K., Kronheim, S. R., March, C. J., Conlon, P. J., Hopp, T. P., Gillis, S., and Urdal, D. L. (1985)J. Exp. Med. 162, 501-515 50. Stylianou, E., ONeill, L. A. J., Rawlinson, L., Edbrooke, M. R., Woo, P., and Saklatvala, J. (1992)J . B i d . Chem. 267, 15836-15841 51. Colotta, F., Re, F., Muzio, M., Bertini, R., Polentarutti, N., Sironi, M., Gin, J. G., Dower, S. K, Sims, J. E., and Mantovani,A. (1993)Science 261,472-475 52. Edwards, C. R. W., Benediktsson, R., Lindsay, R. S., and Seckl, J. R. (1993) Lancet 341,355-357 53. Robinson, B. G., Emanuel, R. L., Frim, D. M., and Majzoub, J. A. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5244-5248 54. Ringler, G. E., Kallen, C. B., and Strauss, J. F., I11 (1989) Endocrinology 124, 1625-1631 55. Jaffe, R. B. (1986) in Reproductive Endocrinology (Yen, S. S. C., and Jaffe, R. B., eds) pp. 758-774, W. B. Saunders Co., Philadelphia 56. Romero, R., Brody, D. T., Oyarzun, E., Mazor, M., Wu, Y. K, Hobbins, J. C., and Durum, S. K. (1989)Am. J . Obstet. Gynecol. 160, 1117-1123 57. Ellis, S.A,, Palmer, M. S., and McMichael, A. J. (1990) J. Immunol. 144, 731-735 58. Defilippi, P., Silengo, L., and Tarone, G. (1992) J. Biol. Chem. 267, 1830318307 59. Hunt, J. S. (1989) J . Reprod. Immunol. 16, 1-17