- Email: [email protected]
Induction and posttranslational expression of cytokines in a first-trimester trophoblast cell line by lipopolysaccharide
Induction and posttranslational expression of cytokines in first-trimester trophoblast cell line by lipopolysaccharide
a
David Martin Svinarich, Phi)," Ornella Maria Bitonti, BS, ~ Roberto Romero, MD," b and Bernard Gonik, MD" Detroit, Michigan, and Bethesda, Maryland OBJECTIVES: The response to infection by human first-trimester trophoblasts is a poorly understood event. This study was undertaken to determine whether first-trimester trophoblasts are capable of responding to an infection stimulus and mediating an immune response. STUDY DI=SI6N: HTR-8/SVneo cells were exposed to lipopolysaccharide (1 ~g/ml) or media alone for either 0, 2, 4, 6, 8, or 24 hours. Northern analysis was conducted by use of a panel of antisense cytokine probes. Enzyme-linked immunosorbent assays specific for either interleukin-la, interleukin-6, interleukin-8, or transforming growth factor-~l were conducted on corresponding cell culture supernatants, and the kinetics of expression were determined. RESULTS: Interleukin-lcq interleukin-6, interleukin-8, and transforming growth factor-~l transcription occurred maximally between 2 and 8 hours of culture in media containing Iipopolysaccharide, with a subsequent diminution of response. Enzyme-linked immunosorbent assay analysis corroborated lipopolysaccharide induction seen at the level of transcription, with significant posttranslational expression of these cytokines being detected between 2 and 24 hours in culture (p < 0.01). CONCLUSIONS: Expression of the proinflammatory cytokines interleukin-lc~, interleukin-6, interleukin-8 and transforming growth factor-~l strongly support the contention that human first-trimester trophoblasts are capable of responding to an infection stimulus and eliciting an immune response through cytokine-based immune signaling. (Am J Obstet Gynecol 1996;175:970-3.)
Key words: Cytokines, trophoblast, infection, immune signaling, lipopolysaccharide
The human placenta is a remarkable endocrine organ that serves both as a selective mechanical harrier and an immunologic barrier against the vertical transmission of infectious agents. In similarity with mononuclear phagocytes, term trophoblasts express or respond to a number of cytokines, including granulocyte-macrophage colonystimulating factor, colony-stimulating factor 1, tumor necrosis factor-et, interleukin-1 (IL-1), interleukin-6 (IL-6), transforming growth factor-~ (TGF-~), and platelet
trating an immunologic defense against infectious agents. However, interpretation of previous data has often been confounded by the presence of contaminating macrophages and resident Haufbauer cells in primary trophoblast cultures) Comparatively little is known about the capacity of first-trimester trophoblasts to respond to an infection during the early gestation of the embryo and what role, if any, these ceils play in mounting an effective immune response. This study was undertaken to determine whether cytokines are expressed in vitro by a first-trimester trophoblast cell line in response to lipopolysaccharide and to profile the kinetics of this expression at the level of transcription and translation. Material and m e t h o d s
Cell culture. HTR-8/SVneo cells were derived from human extravillous trophoblasts. 6 Cells between 18 and 23 passages were grown to confluency in Dulbecco's modified Eagle's m e d i u m - H a m ' s F-12 medium (HyClone Laboratories, Logan, Utah) supplemented with 10% non-heat-inacfivated fetal bovine serum, 1% L-glutamine, 50 U / m l penicillin, and 5 p g / m l streptomycin (Sigma, St. Louis). Cells were maintained at 37 ~ C in an atmosphere of 5% carbon dioxide in 75 cm 2 flasks (Corning, Corning, N.Y). All culture media was tested for the
Svinarich et al,
Volume 175, Number 4, Part 1 Am J Obstet Gynecol
presence of contaminating endotoxin before the addition of lipopolysaccharide by a Limulus assay with a sensitivity of 0.06 to 0.10 n g / m l (Endotect, ICN Biomedicals, Aurora, Ohio). Lipopolysaccharide exposure and culture supernatant collection. Confluent HTR-8/SVneo cell monolayers were washed three times in 4 ml of Dulbecco's modified Eagle's m e d i u m - H a m ' s F-12 medium before culturing u n d e r experimental conditions. Esche*ichia coli serotype 0.55B5 lipopolysaccharide (Sigma) was solubilized in Dulbecco's modified Eagle's m e d i u m - H a m ' s F-12 medium to a concentration of 1 m g / m l and added to culture media at a final concentration of 1 pg/ml. Cells were then exposed to media alone or media containing lipopolysaccharide for either 0, 2, 4, 6, 8, or 24 hours. Culture supernatants used to conduct enzyme-linked immunosorbent assays (ELISA) were obtained from single flasks to minimize variation because of cell number. An 800 pl aliquot of culture supernatant was withdrawn at each of the previously described time intervals and centrifuged at 14,000gfor 5 minutes to remove cellular debris. Supernatams were withdrawn and subsequently stored at -70 ~ C until needed. Ribonucleie acid isolation and Northern analysis. Total ribonucleic acid (RNA) was isolated from transformed trophoblast cell monolayers with TRIzol reagent (Gibco BRL, Gaithersburg, Md.) in accordance with the manufacturer's instructions. A 15 pg aliquot of total RNA from each time point was resolved on a 1% agarose-formaldehyde gel, and residual formaldehyde was removed by two 20-minute washes in 250 ml of diethyl pyrocarbonate-treated water. RNA was electrophoretically transferred onto Magna nylon transfer membranes (Micron Separations, Westboro, Mass.), at a constant 50 V and 250 mA for 16 hours in lx TAE buffer (0.04 m o l / L Trisacetate, 0.001 m o l / L ethylenediaminetetraacetic acid, pH 8.0) with a Trans-Blot apparatus (Bio-Rad Laboratories, Richmond, Calif.). RNA was covalently attached to the transfer membrane by exposure to ultraviolet radiation. Membranes were prehybridized at 65 ~ C for 1 hour in 50 m m o l / L Tris buffer (pH 7.5) containing 1.0 m o l / L sodium chloride, 10% dextran sulfate, 1% sodium dodecyl sulfate, and 100 p g / m l of denatured salmon testes deoxyribonucleic acid. Synthetic oligomeric antisense cytokine and ~actin probes (Integrated DNA Technologies, Coralville, Ia.) were end-labeled with 7-phosphorus 32-labeled deoxycytidine triphosphate and purified through a Sephadex C~25 column (Pharmacia, Piscataway, NJ.). Hybridization was conducted at 65 ~ C for 16 hours in the original solution containing 5 • 10~ c o u n t s / m i n / m l of radiolabeled probe. Northern blots were washed for 30 minutes at 65 ~ C in 2x SSC buffer (3 m o l / L sodium chloride, 0.3 m o l / L sodium citrate, pH 7.0) and 0.1% sodium dodecyl sulfate and then for 5 minutes in fresh solution at room temperature.
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Fig. 1. Northern analysis of cytokine expression in lipopolysaccharide-induced HTR-8/SVneo cells. Total RNA was isolated from cells after 0, 2, 4, 6, 8, and 24 hours of culture in medium containing lipopolysaccharide (1 pg/ml) and subjected to hybridization with either IL-I~ (A), IL-6 (B), IL-8 (C), or TGF-~]I (D) antisense probes. Arrow, Periods of maximal induction. Relative positions of the 18S and 28S ribosomal subfractions are indicated on/eft. Autoradiography was performed for 1 to 10 days at -70 ~ C with Kodak X-OMAT film (Rochester, N.Y) and intensifying screens. Autoradiographs were processed on a Kodak X-OMAT M43A automated processor. Cytokine signals were compared against ~-actin signals obtained from the same membrane to ensure equal RNA loading. ELISA conditions and statistical analysis. IL-I~, IL-6, interleukin-8 (IL-8), and TGF-[~I protein levels were measured with commercial ELISA tests in accordance with the manufacturer's instructions and validated for use with culture supernatant (R&D Systems, Minneapolis). Sensitivities of the assays for IL-10t, IL-6, IL-8, and TGF-~I were 0.2 pg/ml, 0.7 pg/ml, 18.1 pg/ml, and 5.0 pg/ml, respectively. All standards were assayed in duplicate and the values averaged. Absorbance values with a coefficient of variation >12% were not used. All test samples were assayed in duplicate at each time point and the results averaged. Statistical significance between lipopolysaccharide-treated and nonqipopolysaccharidetreated samples was determined with repeated-measures analysis of variance.
972
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October 1996 AmJ Obstet Gyneco[
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Fig. 2. ELISA analysis of cytokine expression in lipopolysaccharide-induced HTR/8/SVneo cells. Cell culture supernatants were analyzed for either IL-10t (A), IL-6 (B), IL-8 (C), or TGF-~I (D) after 0, 2, 4, 6, 8, and 24 hours of culture in either medium alone or medium containing lipopolysaccharide (1 pg/ml). Results
Northern analysis. HTR-8/SVneo total RNA hybridizing with the antisense IL-lc( probe demonstrated a single messenger RNA (mRNA) species of approximately 2430 bases. A very low level of constitutive expression was observed at the 0 hour time point. Maximal levels of expression occurred after 2 hours of lipopolysaccharide exposure and persisted to 24 hours at levels slightly below maximum (Fig. 1, A). Hybridization with the antisense IL-6 probe demonstrated a single mRNA species of approximately 1128 bases. A moderate level of constitutive expression was observed at the 0 hour time point and persisted at essentially the same level to 6 hours, An expression maxima was observed at 8 hours with a subsequent diminishment of signal to near constitutive levels by 24 hours (Fig. 1, B). Hybridization with the antisense IL-8 probe yielded a single mRNA species of approximately 1639 bases. A moderate level of constitutive expression was detected at 0 hours and continued at essentially the same level to 8 hours, when an expression maxima was reached. Signal intensity diminished to levels below maxima but above constitutive levels at 24 hours (Fig. 1, C). Hybridization with the antisense TGF-~I probe demonstrated a single mRNA message of approxi-
mately 2745 bases with a very low constitutive level of expression at 0 hours. Induced levels of TGF-~I were apparent by 2 hours after lipopolysaccbaride exposure, with an expression maxima occurring at approximately 6 hours. Signal intensity diminished to levels below maxima but above constitutive levels at 8 hours and continued to 24 hours (Fig. 1, D). Northern analysis failed to detect the presence of mRNA to the cytokines IL-I[~, interleukin-7, interleukin-10, interleukin-12, interleukin13, or tumor necrosis factor-0~ after prolonged atttoradiographic exposure (data not shown). ELISA analysis. Expression of IL-lc~ protein was detected after approximately 8 hours of lipopolysaccharide exposure and continued to increase in a time-dependent fashion to 24 hours. IL-lc~ was undetectable in noninduced HTR-8/SVneo cells before 24 hours in culture (Fig. 2, A). Induced levels of IL-6 were detectable within 2 hours of culture and increased in a time-dependent manner throughout the course of this experiment. A change in slope by 6 hours after induction correlated with an expression maxima at 6 hours by Northern analysis. Noninduced levels of IL-6 were detectable by approximately 2 hours of culture (Fig. 2, B). Expression of IL-8 became evident in both lipopolysaccharide-induced and nonin-
Volume 175, Number 4, Part 1 AmJ Obstet Gynecol
d u c e d cells by 2 hours in culture and c o n t i n u e d to increase to 24 hours (Fig. 2, C). T h r o u g h o u t the course of dtis e x p e r i m e n t there was a very low level of constitutive IL-6 and IL-8 expression that correlated with N o r t h e r n data. No TGF-[31 protein could be d e t e c t e d in either i n d u c e d or n o n i n d u c e d cells by ELISA before 8 hours in culture. A m o d e s t but significant difference in TGF-[~I levels o c c u r r e d at 24 hours between i n d u c e d and noninduced cells (Fig. 2, D). In all instances i n d u c e d levels of cytokines by 24 hours were significantly greater (p < 0.01) than n o n i n d u c e d levels by 24 hours in culture. Comment
T h e establishment of an effective i m m u n e response requires c o m m u n i c a t i o n a m o n g many cell types and is therefore critically d e p e n d e n t on the ability of these cells to b o t h express and r e s p o n d to a variety of cytokines. Little is currently known about the capacity of h u m a n first-trimester trophoblasts to respond to infections. Previous cytokine studies that used t e r m trophoblasts grown in primary culture have b e e n plagued by the presence of c o n t a m i n a t i n g m a c r o p h a g e s and resident H o f t b a u e r cells. ~ Consequently, these studies were c o n d u c t e d with use of a first-trimester trophoblast cell line. Transcriptional activation of the p r o i n f l a m m a t o r y cytokines IL-lcq IL-6, and IL-8 by lipopolysaccharide, as evidenced by N o r t h e r n analysis, was c o r r o b o r a t e d at the level of translation by ELISA. TGF-[31 is n o t recognized to be directly inducible by lipopolysaccharide. However, H T R - 8 / S V n e o cells did b e c o m e transcriptionally active, and there was a statistically significant level of cytokine expression after culture in the presence of lipopolysaccharide. T h e m i n o r level of induction observed after the addition o f lipopolysaccharide may have o c c u r r e d through the seco n d a r y expression of o t h e r stimulatory cytokines or through an alternate mechanism. This nmltifunctional growth factor has b e e n r e p o r t e d to be expressed at constitutive levels and to m o d u l a t e the invasive properties of first-trimester trophoblasts. 7 This r e p o r t is the first to describe the kinetics of lipopolysaccharide i n d u c t i o n and posttranslafional expression of the cytokines I b l a , IL-6, and IL-8 and the
Svinarich et al.
973
indirect expression of TGF-[~I in a h u m a n first-trimester trophoblast cell line. These data support previous observations by us 4 and others 8 who describe the lipopolysaccharide-inducible expression of IL-10~ and IL-8 and the constitutive expression o f IL-6 in term cytotrophoblasts grown in primary culture. 9 T h e observation that a firsttrimester trophoblast cell line possess a variety of proinflammatory cytokine genes that are both responsive to lipopolysaccharide induction and expressed at the level of translation suggests that differentiation to this p h e n o type occurs early in the o n t o g e n y of the cell. These data strongly support the c o n t e n t i o n that first-trimester trophoblasts are capable of r e s p o n d i n g to an infection stimulus and orchestrating an i m m u n e response through cytokine-based i m m u n e signaling. We thank Charles H. Graham, PhD, for his kind gift of the H T R - 8 / S V n e o cell line.
REFERENCES
1. Guilbert L, Robertson SA, Wegmann TG. The trophoblast as an integral component of a macrophage-cytokine network. Immunol Cell Biol 1993;71:49-57. 2. Yanushpolsky EH, Ozmrk M, Polgar K, Berkowitz RS, HillJA. The effects of cytokines on human chorionic gonadotropin (hCG) production by a trophoblast cell line. J Reprod hnmunol 1993;25:235-47. 3. Dinarello CA. Interleukin-1. Dig Dis Sci 1988;33(suppl 3):2535S. 4. Gonik B, RachmilewitzJ, Hochberg A, Goshen R, deGroot N. Induction of tumor necrosis factor and interleukin-6 mRNA in human cytotrophoblast cells exposed to lipopolysaccharide. Infect Dis Obstet Gynecol 1994;2:3-9. 5. Kliman HJ, Meaddough E. Placental interleukin-8 is predominantly made by villous macrophages, not trophoblasts [abstract]. J Soc Gynecol Invest 1995;2:208. 6. Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N, et al. Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res 1993;206:204-11. 7. Graham CH, Lala PK~ Mechanism of control of trophoblast invasion in situ. J Cell Physiol 1991;148:228-34. 8. Saito S, Kasahara T, Kato Y, Ishihara Y, Ichijo M. Elevation of amniotic fluid interleukin 6 (IL-6), IL-8 and granulocyte colony stimulating factor (G-CSF) in term and preterm parturition. Cytokines 1993;5:81-8. 9. Kameda T, Matsuzaki N, Sawai K, et al. Production of interleukin-6 by normal human trophoblast. Placenta 1990;11:20513.
a
David Martin Svinarich, Phi)," Ornella Maria Bitonti, BS, ~ Roberto Romero, MD," b and Bernard Gonik, MD" Detroit, Michigan, and Bethesda, Maryland OBJECTIVES: The response to infection by human first-trimester trophoblasts is a poorly understood event. This study was undertaken to determine whether first-trimester trophoblasts are capable of responding to an infection stimulus and mediating an immune response. STUDY DI=SI6N: HTR-8/SVneo cells were exposed to lipopolysaccharide (1 ~g/ml) or media alone for either 0, 2, 4, 6, 8, or 24 hours. Northern analysis was conducted by use of a panel of antisense cytokine probes. Enzyme-linked immunosorbent assays specific for either interleukin-la, interleukin-6, interleukin-8, or transforming growth factor-~l were conducted on corresponding cell culture supernatants, and the kinetics of expression were determined. RESULTS: Interleukin-lcq interleukin-6, interleukin-8, and transforming growth factor-~l transcription occurred maximally between 2 and 8 hours of culture in media containing Iipopolysaccharide, with a subsequent diminution of response. Enzyme-linked immunosorbent assay analysis corroborated lipopolysaccharide induction seen at the level of transcription, with significant posttranslational expression of these cytokines being detected between 2 and 24 hours in culture (p < 0.01). CONCLUSIONS: Expression of the proinflammatory cytokines interleukin-lc~, interleukin-6, interleukin-8 and transforming growth factor-~l strongly support the contention that human first-trimester trophoblasts are capable of responding to an infection stimulus and eliciting an immune response through cytokine-based immune signaling. (Am J Obstet Gynecol 1996;175:970-3.)
Key words: Cytokines, trophoblast, infection, immune signaling, lipopolysaccharide
The human placenta is a remarkable endocrine organ that serves both as a selective mechanical harrier and an immunologic barrier against the vertical transmission of infectious agents. In similarity with mononuclear phagocytes, term trophoblasts express or respond to a number of cytokines, including granulocyte-macrophage colonystimulating factor, colony-stimulating factor 1, tumor necrosis factor-et, interleukin-1 (IL-1), interleukin-6 (IL-6), transforming growth factor-~ (TGF-~), and platelet
trating an immunologic defense against infectious agents. However, interpretation of previous data has often been confounded by the presence of contaminating macrophages and resident Haufbauer cells in primary trophoblast cultures) Comparatively little is known about the capacity of first-trimester trophoblasts to respond to an infection during the early gestation of the embryo and what role, if any, these ceils play in mounting an effective immune response. This study was undertaken to determine whether cytokines are expressed in vitro by a first-trimester trophoblast cell line in response to lipopolysaccharide and to profile the kinetics of this expression at the level of transcription and translation. Material and m e t h o d s
Cell culture. HTR-8/SVneo cells were derived from human extravillous trophoblasts. 6 Cells between 18 and 23 passages were grown to confluency in Dulbecco's modified Eagle's m e d i u m - H a m ' s F-12 medium (HyClone Laboratories, Logan, Utah) supplemented with 10% non-heat-inacfivated fetal bovine serum, 1% L-glutamine, 50 U / m l penicillin, and 5 p g / m l streptomycin (Sigma, St. Louis). Cells were maintained at 37 ~ C in an atmosphere of 5% carbon dioxide in 75 cm 2 flasks (Corning, Corning, N.Y). All culture media was tested for the
Svinarich et al,
Volume 175, Number 4, Part 1 Am J Obstet Gynecol
presence of contaminating endotoxin before the addition of lipopolysaccharide by a Limulus assay with a sensitivity of 0.06 to 0.10 n g / m l (Endotect, ICN Biomedicals, Aurora, Ohio). Lipopolysaccharide exposure and culture supernatant collection. Confluent HTR-8/SVneo cell monolayers were washed three times in 4 ml of Dulbecco's modified Eagle's m e d i u m - H a m ' s F-12 medium before culturing u n d e r experimental conditions. Esche*ichia coli serotype 0.55B5 lipopolysaccharide (Sigma) was solubilized in Dulbecco's modified Eagle's m e d i u m - H a m ' s F-12 medium to a concentration of 1 m g / m l and added to culture media at a final concentration of 1 pg/ml. Cells were then exposed to media alone or media containing lipopolysaccharide for either 0, 2, 4, 6, 8, or 24 hours. Culture supernatants used to conduct enzyme-linked immunosorbent assays (ELISA) were obtained from single flasks to minimize variation because of cell number. An 800 pl aliquot of culture supernatant was withdrawn at each of the previously described time intervals and centrifuged at 14,000gfor 5 minutes to remove cellular debris. Supernatams were withdrawn and subsequently stored at -70 ~ C until needed. Ribonucleie acid isolation and Northern analysis. Total ribonucleic acid (RNA) was isolated from transformed trophoblast cell monolayers with TRIzol reagent (Gibco BRL, Gaithersburg, Md.) in accordance with the manufacturer's instructions. A 15 pg aliquot of total RNA from each time point was resolved on a 1% agarose-formaldehyde gel, and residual formaldehyde was removed by two 20-minute washes in 250 ml of diethyl pyrocarbonate-treated water. RNA was electrophoretically transferred onto Magna nylon transfer membranes (Micron Separations, Westboro, Mass.), at a constant 50 V and 250 mA for 16 hours in lx TAE buffer (0.04 m o l / L Trisacetate, 0.001 m o l / L ethylenediaminetetraacetic acid, pH 8.0) with a Trans-Blot apparatus (Bio-Rad Laboratories, Richmond, Calif.). RNA was covalently attached to the transfer membrane by exposure to ultraviolet radiation. Membranes were prehybridized at 65 ~ C for 1 hour in 50 m m o l / L Tris buffer (pH 7.5) containing 1.0 m o l / L sodium chloride, 10% dextran sulfate, 1% sodium dodecyl sulfate, and 100 p g / m l of denatured salmon testes deoxyribonucleic acid. Synthetic oligomeric antisense cytokine and ~actin probes (Integrated DNA Technologies, Coralville, Ia.) were end-labeled with 7-phosphorus 32-labeled deoxycytidine triphosphate and purified through a Sephadex C~25 column (Pharmacia, Piscataway, NJ.). Hybridization was conducted at 65 ~ C for 16 hours in the original solution containing 5 • 10~ c o u n t s / m i n / m l of radiolabeled probe. Northern blots were washed for 30 minutes at 65 ~ C in 2x SSC buffer (3 m o l / L sodium chloride, 0.3 m o l / L sodium citrate, pH 7.0) and 0.1% sodium dodecyl sulfate and then for 5 minutes in fresh solution at room temperature.
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Fig. 1. Northern analysis of cytokine expression in lipopolysaccharide-induced HTR-8/SVneo cells. Total RNA was isolated from cells after 0, 2, 4, 6, 8, and 24 hours of culture in medium containing lipopolysaccharide (1 pg/ml) and subjected to hybridization with either IL-I~ (A), IL-6 (B), IL-8 (C), or TGF-~]I (D) antisense probes. Arrow, Periods of maximal induction. Relative positions of the 18S and 28S ribosomal subfractions are indicated on/eft. Autoradiography was performed for 1 to 10 days at -70 ~ C with Kodak X-OMAT film (Rochester, N.Y) and intensifying screens. Autoradiographs were processed on a Kodak X-OMAT M43A automated processor. Cytokine signals were compared against ~-actin signals obtained from the same membrane to ensure equal RNA loading. ELISA conditions and statistical analysis. IL-I~, IL-6, interleukin-8 (IL-8), and TGF-[~I protein levels were measured with commercial ELISA tests in accordance with the manufacturer's instructions and validated for use with culture supernatant (R&D Systems, Minneapolis). Sensitivities of the assays for IL-10t, IL-6, IL-8, and TGF-~I were 0.2 pg/ml, 0.7 pg/ml, 18.1 pg/ml, and 5.0 pg/ml, respectively. All standards were assayed in duplicate and the values averaged. Absorbance values with a coefficient of variation >12% were not used. All test samples were assayed in duplicate at each time point and the results averaged. Statistical significance between lipopolysaccharide-treated and nonqipopolysaccharidetreated samples was determined with repeated-measures analysis of variance.
972
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October 1996 AmJ Obstet Gyneco[
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Fig. 2. ELISA analysis of cytokine expression in lipopolysaccharide-induced HTR/8/SVneo cells. Cell culture supernatants were analyzed for either IL-10t (A), IL-6 (B), IL-8 (C), or TGF-~I (D) after 0, 2, 4, 6, 8, and 24 hours of culture in either medium alone or medium containing lipopolysaccharide (1 pg/ml). Results
Northern analysis. HTR-8/SVneo total RNA hybridizing with the antisense IL-lc( probe demonstrated a single messenger RNA (mRNA) species of approximately 2430 bases. A very low level of constitutive expression was observed at the 0 hour time point. Maximal levels of expression occurred after 2 hours of lipopolysaccharide exposure and persisted to 24 hours at levels slightly below maximum (Fig. 1, A). Hybridization with the antisense IL-6 probe demonstrated a single mRNA species of approximately 1128 bases. A moderate level of constitutive expression was observed at the 0 hour time point and persisted at essentially the same level to 6 hours, An expression maxima was observed at 8 hours with a subsequent diminishment of signal to near constitutive levels by 24 hours (Fig. 1, B). Hybridization with the antisense IL-8 probe yielded a single mRNA species of approximately 1639 bases. A moderate level of constitutive expression was detected at 0 hours and continued at essentially the same level to 8 hours, when an expression maxima was reached. Signal intensity diminished to levels below maxima but above constitutive levels at 24 hours (Fig. 1, C). Hybridization with the antisense TGF-~I probe demonstrated a single mRNA message of approxi-
mately 2745 bases with a very low constitutive level of expression at 0 hours. Induced levels of TGF-~I were apparent by 2 hours after lipopolysaccbaride exposure, with an expression maxima occurring at approximately 6 hours. Signal intensity diminished to levels below maxima but above constitutive levels at 8 hours and continued to 24 hours (Fig. 1, D). Northern analysis failed to detect the presence of mRNA to the cytokines IL-I[~, interleukin-7, interleukin-10, interleukin-12, interleukin13, or tumor necrosis factor-0~ after prolonged atttoradiographic exposure (data not shown). ELISA analysis. Expression of IL-lc~ protein was detected after approximately 8 hours of lipopolysaccharide exposure and continued to increase in a time-dependent fashion to 24 hours. IL-lc~ was undetectable in noninduced HTR-8/SVneo cells before 24 hours in culture (Fig. 2, A). Induced levels of IL-6 were detectable within 2 hours of culture and increased in a time-dependent manner throughout the course of this experiment. A change in slope by 6 hours after induction correlated with an expression maxima at 6 hours by Northern analysis. Noninduced levels of IL-6 were detectable by approximately 2 hours of culture (Fig. 2, B). Expression of IL-8 became evident in both lipopolysaccharide-induced and nonin-
Volume 175, Number 4, Part 1 AmJ Obstet Gynecol
d u c e d cells by 2 hours in culture and c o n t i n u e d to increase to 24 hours (Fig. 2, C). T h r o u g h o u t the course of dtis e x p e r i m e n t there was a very low level of constitutive IL-6 and IL-8 expression that correlated with N o r t h e r n data. No TGF-[31 protein could be d e t e c t e d in either i n d u c e d or n o n i n d u c e d cells by ELISA before 8 hours in culture. A m o d e s t but significant difference in TGF-[~I levels o c c u r r e d at 24 hours between i n d u c e d and noninduced cells (Fig. 2, D). In all instances i n d u c e d levels of cytokines by 24 hours were significantly greater (p < 0.01) than n o n i n d u c e d levels by 24 hours in culture. Comment
T h e establishment of an effective i m m u n e response requires c o m m u n i c a t i o n a m o n g many cell types and is therefore critically d e p e n d e n t on the ability of these cells to b o t h express and r e s p o n d to a variety of cytokines. Little is currently known about the capacity of h u m a n first-trimester trophoblasts to respond to infections. Previous cytokine studies that used t e r m trophoblasts grown in primary culture have b e e n plagued by the presence of c o n t a m i n a t i n g m a c r o p h a g e s and resident H o f t b a u e r cells. ~ Consequently, these studies were c o n d u c t e d with use of a first-trimester trophoblast cell line. Transcriptional activation of the p r o i n f l a m m a t o r y cytokines IL-lcq IL-6, and IL-8 by lipopolysaccharide, as evidenced by N o r t h e r n analysis, was c o r r o b o r a t e d at the level of translation by ELISA. TGF-[31 is n o t recognized to be directly inducible by lipopolysaccharide. However, H T R - 8 / S V n e o cells did b e c o m e transcriptionally active, and there was a statistically significant level of cytokine expression after culture in the presence of lipopolysaccharide. T h e m i n o r level of induction observed after the addition o f lipopolysaccharide may have o c c u r r e d through the seco n d a r y expression of o t h e r stimulatory cytokines or through an alternate mechanism. This nmltifunctional growth factor has b e e n r e p o r t e d to be expressed at constitutive levels and to m o d u l a t e the invasive properties of first-trimester trophoblasts. 7 This r e p o r t is the first to describe the kinetics of lipopolysaccharide i n d u c t i o n and posttranslafional expression of the cytokines I b l a , IL-6, and IL-8 and the
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indirect expression of TGF-[~I in a h u m a n first-trimester trophoblast cell line. These data support previous observations by us 4 and others 8 who describe the lipopolysaccharide-inducible expression of IL-10~ and IL-8 and the constitutive expression o f IL-6 in term cytotrophoblasts grown in primary culture. 9 T h e observation that a firsttrimester trophoblast cell line possess a variety of proinflammatory cytokine genes that are both responsive to lipopolysaccharide induction and expressed at the level of translation suggests that differentiation to this p h e n o type occurs early in the o n t o g e n y of the cell. These data strongly support the c o n t e n t i o n that first-trimester trophoblasts are capable of r e s p o n d i n g to an infection stimulus and orchestrating an i m m u n e response through cytokine-based i m m u n e signaling. We thank Charles H. Graham, PhD, for his kind gift of the H T R - 8 / S V n e o cell line.
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1. Guilbert L, Robertson SA, Wegmann TG. The trophoblast as an integral component of a macrophage-cytokine network. Immunol Cell Biol 1993;71:49-57. 2. Yanushpolsky EH, Ozmrk M, Polgar K, Berkowitz RS, HillJA. The effects of cytokines on human chorionic gonadotropin (hCG) production by a trophoblast cell line. J Reprod hnmunol 1993;25:235-47. 3. Dinarello CA. Interleukin-1. Dig Dis Sci 1988;33(suppl 3):2535S. 4. Gonik B, RachmilewitzJ, Hochberg A, Goshen R, deGroot N. Induction of tumor necrosis factor and interleukin-6 mRNA in human cytotrophoblast cells exposed to lipopolysaccharide. Infect Dis Obstet Gynecol 1994;2:3-9. 5. Kliman HJ, Meaddough E. Placental interleukin-8 is predominantly made by villous macrophages, not trophoblasts [abstract]. J Soc Gynecol Invest 1995;2:208. 6. Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N, et al. Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res 1993;206:204-11. 7. Graham CH, Lala PK~ Mechanism of control of trophoblast invasion in situ. J Cell Physiol 1991;148:228-34. 8. Saito S, Kasahara T, Kato Y, Ishihara Y, Ichijo M. Elevation of amniotic fluid interleukin 6 (IL-6), IL-8 and granulocyte colony stimulating factor (G-CSF) in term and preterm parturition. Cytokines 1993;5:81-8. 9. Kameda T, Matsuzaki N, Sawai K, et al. Production of interleukin-6 by normal human trophoblast. Placenta 1990;11:20513.