Preeclampsia-Related Inflammatory Cytokines Regulate Interleukin-6 Expression in Human Decidual Cells
2008; Elsevier BV; Volume: 172; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.070629
ISSN1525-2191
AutoresCharles J. Lockwood, Chih‐Feng Yen, Murat Başar, Umit A. Kayisli, Maritza Martel, Irina A. Buhimschi, Catalin S. Buhimschi, S. Joseph Huang, Graciela Krikun, Frederick Schatz,
Tópico(s)Preterm Birth and Chorioamnionitis
ResumoPreeclampsia, a common pregnancy disorder associated with an increase in systemic inflammation, is the leading cause of maternal and fetal morbidity and mortality throughout the world. It is associated with shallow extravillous trophoblast invasion of the decidua, leading to uteroplacental blood flow that is inadequate for the developing fetal-placental unit. In preeclamptic women, interleukin-6 (IL-6) levels in plasma, but not placenta, are elevated, prompting evaluation of the decidua as a potential source of this excess, circulating IL-6. The current study found significantly higher immunohistochemical staining for IL-6 in decidual cells from preeclamptic versus preterm, gestational age-matched control placentas. Pro-inflammatory cytokines associated with the genesis of preeclampsia (i.e., tumor necrosis factor-α and interleukin-1β) enhanced IL-6 mRNA levels and increased secreted IL-6 levels in first trimester leukocyte-free decidual cell incubations, as measured by real time quantitative RT-PCR, ELISA, and Western blotting. Therefore, decidual cell-derived IL-6 may contribute to excess circulating IL-6 levels that can promote both endothelial cell dysfunction (and subsequent vascular dysfunction) and the pathogenesis of preeclampsia whereas locally elevated IL-6 levels may contribute to an excess of decidual macrophages implicated in shallow extravillous trophoblast invasion of the decidua. Preeclampsia, a common pregnancy disorder associated with an increase in systemic inflammation, is the leading cause of maternal and fetal morbidity and mortality throughout the world. It is associated with shallow extravillous trophoblast invasion of the decidua, leading to uteroplacental blood flow that is inadequate for the developing fetal-placental unit. In preeclamptic women, interleukin-6 (IL-6) levels in plasma, but not placenta, are elevated, prompting evaluation of the decidua as a potential source of this excess, circulating IL-6. The current study found significantly higher immunohistochemical staining for IL-6 in decidual cells from preeclamptic versus preterm, gestational age-matched control placentas. Pro-inflammatory cytokines associated with the genesis of preeclampsia (i.e., tumor necrosis factor-α and interleukin-1β) enhanced IL-6 mRNA levels and increased secreted IL-6 levels in first trimester leukocyte-free decidual cell incubations, as measured by real time quantitative RT-PCR, ELISA, and Western blotting. Therefore, decidual cell-derived IL-6 may contribute to excess circulating IL-6 levels that can promote both endothelial cell dysfunction (and subsequent vascular dysfunction) and the pathogenesis of preeclampsia whereas locally elevated IL-6 levels may contribute to an excess of decidual macrophages implicated in shallow extravillous trophoblast invasion of the decidua. 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Biopsies of decidua basalis used for immunohistochemistry obtained from placentas of both preeclamptic and gestational age-matched controls, were formalin-fixed and paraffin-embedded under approval of the Yale University School of Medicine Human Investigation Committee. Table 1 provides relevant clinical details of the patients and controls. Patients with preeclampsia (n = 10) were diagnosed according to standard criteria,42ACOG Committee on Obstetric Practice ACOG practice bulletin. Diagnosis and management of preeclampsia and eclampsia. Number 33, January 2002. American College of Obstetricians and Gynecologists.Obstet Gynecol. 2002; 99: 159-167Crossref PubMed Google Scholar with nine of the patients meeting the criteria for severe preeclampsia with either a systolic blood pressure >160 mmHg or a diastolic blood pressure >110 mmHg on two occasions 6 hours apart, with the presence of significant proteinuria >5 g in a 24-hour urine collection. Placental specimens were obtained from cesarean delivery without labor in all preeclamptic patients or gestational age-matched controls with idiopathic preterm labor without signs of preeclampsia (n = 10). Control placental specimens were obtained after either cesarean or vaginal delivery and none displayed any clinical or histological evidence of chorioamnionitis or chronic villitis.Table 1Characteristics of the Women Who Provided Placental SamplesVariablePreeclampsia (n = 10)Preterm control (n = 10)P valueAge, years*Data presented as mean ± SEM and analyzed by Student's t-test.31 ± 324 ± 30.065Nulliparity†Data presented as n (%) and analyzed by Fisher's exact test.5 (50)5 (50)1.000Gestational age, weeks*Data presented as mean ± SEM and analyzed by Student's t-test.29.7 ± 1.230.5 ± 1.00.605Systolic blood pressure, mmHg*Data presented as mean ± SEM and analyzed by Student's t-test.172 ± 7117 ± 6<0.001Diastolic blood pressure, mmHg*Data presented as mean ± SEM and analyzed by Student's t-test.102 ± 262 ± 3<0.001Dipstick protein‡Data presented as median [range] and analyzed by Mann-Whitney test.4 [2 to 4]0 [0 to 0]<0.00124-hour protein, g5 [0.7 to 7.2]NANAIUGR†Data presented as n (%) and analyzed by Fisher's exact test.3 (30)0 (0)0.211HELLP syndrome†Data presented as n (%) and analyzed by Fisher's exact test.3 (30)0 (0)0.211PPROM†Data presented as n (%) and analyzed by Fisher's exact test.0 (0)5 (50)0.033Birth weight, g*Data presented as mean ± SEM and analyzed by Student's t-test.1077 ± 1391532 ± 2020.085Cesarean delivery†Data presented as n (%) and analyzed by Fisher's exact test.8 (80)3 (30)0.070Histological chorioamnionitis stages II to III†Data presented as n (%) and analyzed by Fisher's exact test.0 (0)0 (0)1.000PPROM, preterm premature rupture of the membranes; IUGR, intrauterine growth restriction; HELLP, syndrome of hemolysis, elevated liver enzymes, and low platelets.* Data presented as mean ± SEM and analyzed by Student's t-test.† Data presented as n (%) and analyzed by Fisher's exact test.‡ Data presented as median [range] and analyzed by Mann-Whitney test. Open table in a new tab PPROM, preterm premature rupture of the membranes; IUGR, intrauterine growth restriction; HELLP, syndrome of hemolysis, elevated liver enzymes, and low platelets. First-trimester decidual specimens were obtained from nine elective terminations between 8 and 12 weeks of gestation under Institutional Review Board approval at Bellevue Hospital, New York, NY. After separating the decidua from the amnio-chorion, a small portion of each specimen was formalin-fixed and paraffin-embedded then histologically examined for signs of underlying acute and chronic inflammation. The remainder was used for decidual cell isolation. Five-μm sections of paraffin-embedded samples were deparaffinized in xylene and rehydrated in a graded ethanol series. The slides were then boiled in citrate buffer (10 mmol/L, pH 6.0) for 15 minutes for antigen retrieval. Sections were immersed in 3% hydrogen peroxide (in 50% methanol/50% distilled water) for 15 minutes to block endogenous peroxidase then incubated in a humidified chamber with 5% blocking horse serum in Tris-buffered saline (TBS) (Lab-Vision, Fremont, CA) for 30 minutes at room temperature. After removing excess serum, the serial sections were incubated with either mouse monoclonal anti-human IL-6 antibody (10 μg/ml) in 1% blocking horse serum in TBS (R&D Systems, Minneapolis, MN) or mouse monoclonal anti-vimentin antibody or anti-cytokeratin antibody (1:100 dilution in TBS) (DakoCytomation, Carpinteria, CA) overnight at 4°C in a humidified chamber. For negative controls, normal mouse IgG isotypes were used at the same concentrations as the primary antibodies. The sections were washed three times for 5 minutes in TBS then treated with biotinylated horse anti-mouse secondary antibody (Vector Laboratories, Burlingame, CA) at a 1:400 dilution for 30 minutes at room temperature. The antigen-antibody complex was detected with an avidin-biotin-peroxidase kit (Vector Laboratories). Diaminobenzidine (3,3-diaminobenzidine tetrahydrochloride dehydrate) (Vector Laboratories) was used as the chromogen, and sections were counterstained with hematoxylin and mounted with Permount (Fisher Chemicals, Springfield, NJ) on glass slides. The intensity of IL-6 immunostaining was evaluated semiquantitatively using the following categories: 0 (no staining), 1+ (weak, but detectable, staining), 2+ (moderate or distinct staining), and 3+ (intense staining). For each specimen an HSCORE value was derived by calculating the sum of the percentage of cells that stained in each intensity category and multiplying that value by the weighted intensity of the staining, using the formula HSCORE = Pi (i + l), where i represents the intensity score, and Pi is the corresponding percentage of cells.43Kayisli O Kayisli UA Luleci G Arici A In vivo and in vitro regulation of Akt activation in human endometrial cells is estrogen dependent.Biol Reprod. 2004; 71: 714-721Crossref PubMed Scopus (83) Google Scholar In each slide, five different areas and 100 cells per area were evaluated microscopically with a ×40 objective magnification. The percentage of cells at each intensity within these areas was determined at different times by two investigators blinded to the source of the samples, and the average score was used. Digestion of minced tissues with 0.1% collagenase type IV and 0.01% DNase in RPMI containing 20 μg/ml of penicillin/streptomycin and 1 μl/ml of fungizone) (Invitrogen, Carlsbad, CA) in a 37°C shaking water bath for 30 minutes was followed by washing with sterile phosphate-buffered saline (PBS) for three times and then subjected to consecutive filtration through 100-μm, 70-μm, and 40-μm Millipore filters (Millipore Corp., Bedford, MA). Cells were resuspended in RPMI, grown to confluence on polystyrene tissue culture dishes, harvested using trypsin/ethylenediaminetetraacetic acid, and analyzed by flow cytometric analysis with anti-CD45 and anti-CD14 monoclonal antibodies (BD Pharmingen, San Diego, CA) to monitor the presence of leukocytes after each passage. After three to four passages, cell cultures were found to be leukocyte-free (<1%). Cultured decidual cells were vimentin-positive and cytokeratin-negative and displayed decidualization-related morphological and biochemical changes during incubation with a progestin. Decidualization-related biochemical changes included enhanced expression of prolactin and plasminogen activator inhibitor-1 (PAI-1) and inhibited expression of interstitial collagenase and stromelysin-1 (data not shown). Cell aliquots were frozen in fetal calf serum/dimethyl sulfoxide (9:1) (Sigma-Aldrich, St. Louis, MO) and stored in liquid nitrogen. Thawed cells were incubated in basal medium [a phenol red-free 1:1 v/v mix of Dulbecco's modified Eagle's medium (Invitrogen) and Ham's F-12 (Flow Labs, Rockville, MD), with 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml fungizone] supplemented with 10% charcoal-stripped calf serum. After two additional passages, confluent cultures were incubated in parallel in BMS containing either 0.1% ethanol, as vehicle control or 10−8 mol/L estradiol (E2) with or without 10−7 mol/L MPA (Sigma-Aldrich), which was used in place of progesterone because of its greater stability in culture.44Arici A Marshburn PB MacDonald PC Dombrowski RA Progesterone metabolism in human endometrial stromal and gland cells in culture.Steroids. 1999; 64: 530-534Crossref PubMed Scopus (34) Google Scholar The use of E2 as the control incubation for E2 plus MPA was prompted by elevated circulating E2 and progesterone levels during gestation. After 7 days, the cultures were washed twice with Hanks' balanced salt solution to remove residual serum elements. The cultures were switched to a defined medium (DM) consisting of BM plus ITS+ (Collaborative Research, Waltham, MA), 5 μmol/L FeSO4, 50 μmol/L ZnSO4, 1 nmol/L CuSO4, 20 nmol/L Na2SeO3, trace elements (Invitrogen), 50 μg/ml ascorbic acid (Sigma-Aldrich), and 50 ng/ml epidermal growth factor (Becton-Dickinson, Bedford, MA) with either vehicle or steroids or 0.01 to 10 ng/ml of IL-1β or TNF-α (R&D Systems). After the test period, cells were harvested by scraping into ice-cold PBS, pelleted, and extracted in ice-cold lysis buffer. Conditioned medium supernatants and cell lysates were stored at −70°C. Total RNA was extracted from parallel incubations with Tri Reagent (Sigma-Aldrich). Total cell protein levels were measured by a modified Lowry assay (Bio-Rad Laboratories, Inc., Hercules, CA). An ELISA kit from R&D Systems measured IL-6 levels in the cell-conditioned DM according to the manufacturer's instructions. The sensitivity of the ELISA is 0.7 pg/ml with intra-assay and interassay coefficients of variation of 3.1% and 2.7%, respectively. Western blot analysis was performed on concentrated conditioned DM supernatants, which were diluted 1:1 in Laemmli sample buffer and then boiled for 3 minutes. The samples were subjected to electrophoresis on a 10 to 20% sodium dodecyl sulfate polyacrylamide linear
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