Regulation of Monocyte Chemoattractant Protein-1 Expression by Tumor Necrosis Factor-α and Interleukin-1β in First Trimester Human Decidual Cells
2006; Elsevier BV; Volume: 168; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2006.050082
ISSN1525-2191
AutoresCharles J. Lockwood, Paul Matta, Graciela Krikun, Louise A. Koopman, Rachel Masch, Paolo Toti, Felice Arcuri, Se-Te Joseph Huang, Edmund F. Funai, Frederick Schatz,
Tópico(s)Preterm Birth and Chorioamnionitis
ResumoThe current study describes a statistically significant increase in macrophages (CD68-positive cells) in the decidua of preeclamptic patients. To elucidate the regulation of this monocyte infiltration, expression of monocyte chemoattractant protein-1 (MCP-1) was assessed in leukocyte-free first trimester decidual cells. Confluent decidual cells were primed for 7 days in either estradiol or estradiol plus medroxyprogesterone acetate to mimic the decidualizing steroidal milieu of the luteal phase and early pregnancy. The medium was exchanged for a serum-free defined medium containing corresponding steroids +/− tumor necrosis factor (TNF)-α or interleukin (IL)-1β. After 24 hours, enzyme-linked immunosorbent assay measurements indicated that the addition of medroxyprogesterone acetate did not affect MCP-1 output, whereas 10 ng/ml of TNF-α or IL-1β increased output by 83.5-fold ± 20.6 and 103.1-fold ± 14.7, respectively (mean ± SEM, n = 8, P < 0.05). Concentration-response comparisons revealed that even 0.01 ng/ml of TNF-α or IL-1β elevated MCP-1 output by more than 15-fold. Western blotting confirmed the enzyme-linked immunosorbent assay results, and quantitative reverse transcriptase-polymerase chain reaction confirmed corresponding effects on MCP-1 mRNA levels. The current study demonstrates that TNF-α and IL-1β enhance MCP-1 in first trimester decidua. This finding suggests a mechanism by which recruitment of excess macrophages to the decidua impairs endovascular trophoblast invasion, the primary placental defect of preeclampsia. The current study describes a statistically significant increase in macrophages (CD68-positive cells) in the decidua of preeclamptic patients. To elucidate the regulation of this monocyte infiltration, expression of monocyte chemoattractant protein-1 (MCP-1) was assessed in leukocyte-free first trimester decidual cells. Confluent decidual cells were primed for 7 days in either estradiol or estradiol plus medroxyprogesterone acetate to mimic the decidualizing steroidal milieu of the luteal phase and early pregnancy. The medium was exchanged for a serum-free defined medium containing corresponding steroids +/− tumor necrosis factor (TNF)-α or interleukin (IL)-1β. After 24 hours, enzyme-linked immunosorbent assay measurements indicated that the addition of medroxyprogesterone acetate did not affect MCP-1 output, whereas 10 ng/ml of TNF-α or IL-1β increased output by 83.5-fold ± 20.6 and 103.1-fold ± 14.7, respectively (mean ± SEM, n = 8, P < 0.05). Concentration-response comparisons revealed that even 0.01 ng/ml of TNF-α or IL-1β elevated MCP-1 output by more than 15-fold. Western blotting confirmed the enzyme-linked immunosorbent assay results, and quantitative reverse transcriptase-polymerase chain reaction confirmed corresponding effects on MCP-1 mRNA levels. The current study demonstrates that TNF-α and IL-1β enhance MCP-1 in first trimester decidua. This finding suggests a mechanism by which recruitment of excess macrophages to the decidua impairs endovascular trophoblast invasion, the primary placental defect of preeclampsia. 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Several lines of evidence support an association between excess decidual macrophage infiltration and failure of endovascular trophoblast invasion.3Kaufmann P Black S Huppertz B Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia.Biol Reprod. 2003; 69: 1-7Crossref PubMed Scopus (901) Google Scholar, 11Reister F Frank HG Heyl W Kosanke G Huppertz B Schroder W Kaufmann P Rath W The distribution of macrophages in spiral arteries of the placental bed in pre-eclampsia differs from that in healthy patients.Placenta. 1999; 20: 229-233Abstract Full Text PDF PubMed Scopus (125) Google Scholar, 12Reister F Frank HG Kingdom JC Heyl W Kaufmann P Rath W Huppertz B Macrophage-induced apoptosis limits endovascular trophoblast invasion in the uterine wall of preeclamptic women.Lab Invest. 2001; 81: 1143-1152Crossref PubMed Scopus (250) Google Scholar Paradoxically, there are also reports that macrophage numbers in the decidua are unchanged or decreased in preeclampsia.31Burk MR Troeger C Brinkhaus R Holzgreve W Hahn S Severely reduced presence of tissue macrophages in the basal plate of pre-eclamptic placentae.Placenta. 2001; 22: 309-316Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 32Redline RW Macrophages in the basal plate of pre-eclamptic placentae.Placenta. 2001; 22: 890-893Abstract Full Text PDF PubMed Scopus (1) Google Scholar Therefore, we first conducted immunohistochemical staining for the presence of macrophages in decidua obtained from preeclamptic versus unaffected patients. After confirming that preeclamptic placental beds contain excess decidual macrophages, we sought to identify regulators of such macrophage infiltration by assessing the effects of TNF-α and IL-1β on MCP-1 expression in leukocyte-free first trimester decidual cells. The integral role played by progesterone in inducing and maintaining decidualization prompted us to determine whether progestin modulates cytokine effects on MCP-1 expression in the cultured decidual cells. Seven placentas were obtained from preeclamptic women, defined as having elevated blood pressure (>140/90 mmHg) and proteinuria (>+1) on two occasions 6 hours apart after 20 weeks of gestation. Seven control placentas were obtained after uncomplicated pregnancies. All cases and controls were at term and delivered by cesarean section. The study was approved by the local ethics committee (University of Siena, Siena, Italy), and informed consent was obtained from all women. Placentas were obtained immediately after delivery and several (10 mm × 10 mm) full-thickness blocks were cut and either fixed in 10% buffered neutral formalin and embedded in paraffin or frozen in Tissue-Tek OCT (Sakura Finetek, Torrance, CA) medium in liquid nitrogen and stored at −80°C. For each specimen, the block most representative of the maternal decidua was selected for immunohistochemistry. Decidual specimens from elective terminations between 6 and 12 weeks of gestation were obtained with the approval of the Institutional Review Board of New York University Medical Center-Bellevue Hospital, New York, NY. A small portion of each specimen was formalin-fixed and paraffin-embedded then examined histologically for signs of underlying acute and chronic inflammation. The remainder was used for decidual cell isolation. Sections (4 μm) of paraffin-embedded placental tissues were cut, deparaffinized, rehydrated, and washed in Tris-buffered saline [20 mmol/L Tris-HCl, 150 mmol/L NaCl (pH 7.6)]. TBS was used for all subsequent washes and for dilution of the antibody. Antigen retrieval was performed by incubating sections in sodium citrate buffer (10 mmol/L, pH 6.0) in a microwave oven at 750 W for 5 minutes. Sections were subsequently rinsed in 3% hydrogen peroxide to block endogenous peroxidase and incubated for 1 hour at room temperature with a monoclonal antibody against the macrophage marker CD68 (Dakopatts, Carpinteria, CA). Immunostaining was visualized using the avidin-biotin peroxidase complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO) as chromogen substrate. Light hematoxylin stain was used for nuclear counterstaining. Sections of selected specimens were stained for vimentin with a monoclonal antibody (Dakopatts) following the procedure detailed above. Negative controls for each tissue section were prepared by substituting the primary antibody with the corresponding preimmune serum. The CD68-positive cells in the decidua were evaluated by two independent observers using a semiquantitative method in accordance with the following scoring system: 0, absence of positive cells in the decidua; 1, presence of few isolated cells; 2, numerous isolated cells; 3, numerous positive cells, either isolated and/or grouped in clusters. Serial 4-μm cryostat sections of term placentas were mounted on uncoated glass slides, fixed in 70% ethanol, and stained with the HistoGene LCM frozen section staining kit (Arcturus, Mountain View, CA). Pure decidual cells were isolated using a PixCell II LCM system equipped with an Olympus microscope (Arcturus). Captured microdissected decidual cells from two different slides were pooled for subsequent analyses. Tissues were minced and digested with 0.1% collagenase type IV, as well as 0.01% DNase in RPMI containing 20 μg/ml penicillin/streptomycin and 1 μl/ml fungizone (Invitrogen, Grand Island, NY) in a 37°C shaking water bath for 30 minutes. After washing with sterile phosphate-buffered saline (PBS) the digestate was washed three times and subjected to consecutive filtration through 100-μm, 70-μm, and 40-μm Millipore filters (Bedford, MA). Cells were then resuspended in RPMI and seeded on polystyrene tissue culture dishes. Cells were harvested using trypsin/ethylenediaminetetraacetic acid and analyzed by flow cytometric analysis with anti-CD45 and anti-CD14 monoclonal antibodies (mAbs) (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%). Cell aliquots were then frozen in fetal calf serum/dimethyl sulfoxide (9:1) (Sigma-Aldrich) 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, and 0.25 μg/ml fungizone supplemented with 10% charcoal-stripped calf serum (BMS). After two more passages, confluent cultures were incubated in parallel in BMS containing either 10−8 mol/L estradiol (E2) or E2 plus 10−7 mol/L medroxyprogesterone acetate (MPA) (Sigma-Aldrich). After 7 days, the cultures were washed twice with Hanks' balanced salt solution to remove residual serum. The cultures were then switched to a defined medium (DM) consisting of basal medium plus ITS+ (Collaborative Research, Waltham, MA), 5 μmol/L FeSO4, 50 μmol/L ZnSO4, 1 nmol/L CuSO4, 20 nmol/L Na2SeO3, and trace elements (Invitrogen) as well as 50 μg/ml of ascorbic acid (Sigma-Aldrich) and 50 ng/ml of epidermal growth factor (Becton-Dickinson, Bedford, MA) with either vehicle control (0.1% ethanol) or steroids added +/− IL-1β or TNF-α (R&D Systems, Minneapolis, MN). 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 cell protein levels were measured by the Bio-Rad assay (Bio-Rad Laboratories, Inc., Hercules, CA). A commercial enzyme-linked immunosorbent assay (ELISA) kit was used to measure immunoreactive levels of MCP-1 in the cell-conditioned medium according to instructions provided by the manufacturer (R&D Systems). The ELISA assay has a sensitivity of 5.0 pg/ml, and intra- and interassay coefficients of variation of 5.0% and 5.1%, respectively. Western blot analysis was conducted on supernatants of conditioned medium concentrated using Microcon filter devices (Millipore) and then diluted 1:1 in reducing sample buffer composed of Laemmli sample buffer and 2-mercaptoethanol (Bio-Rad) and boiled for 3 minutes. The prepared media was subjected to electrophoresis on a 10 to 20% sodium dodecyl sulfate-polyacrylamide linear gradient gel (Bio-Rad). The gel was electroblotted onto a 0.2-μm nitrocellulose membrane (Bio-Rad). After transfer, the membrane was blocked overnight in PBS with 4% bovine serum albumin and then incubated for 2 hours in 1.5 μg/ml of a mouse anti-human MCP-1 monoclonal antibody (R&D Systems) diluted in PBS with 1% casein. Membranes were rinsed in PBS and 0.2% Tween 20 before and after incubation with horseradish peroxidase-conjugated anti-mouse IgG (ICN Biomedicals, Aurora, OH). Chemiluminescence was detected with ECL reagents (Perkin Elmer Life Sciences, Boston, MA) and audioradiography film (Amersham Pharmacia, Buckinghamshire, UK) according to the instructions provided by the manufacturer. Total RNA of cultured cells was extracted with Tri Reagent (Sigma-Aldrich). Total RNA from the LCM-captured cells was extracted using the RNeasy micro kit (Qiagen Inc., Valencia, CA) according to the manufacturer's recommendation. RNA was subjected to RT-PCR with a kit from Invitrogen (Carlsbad, CA) on an Eppendorf Mastercycler (Eppendorf, Westbury, NY). For each RNA specimen, a negative control was prepared by omitting the reverse transcriptase. To perform quantitative real-time RT-PCR, reverse transcription was initially performed with AMV reverse transcriptase (Invitrogen). A quantitative standard curve was created between 500 pg to 250 ng of cDNA with a Roche Light Cycler (Roche, Indianapolis, IN) by monitoring increasing fluorescence of PCR products during amplification. On establishing the standard curve, quantitation of the unknowns was determined with the Roche Light Cycler and adjusted to the quantitative expression of β-actin from the corresponding unknowns. Melting curve analysis determined the specificity of the amplified products and the absence of primer-dimer formation. All products obtained yielded correct melting temperatures. The following primers were synthesized and gel-purified at the Yale DNA Synthesis Laboratory, Critical Technologies. For MCP-1 mRNA detection in LCM-isolated cells, sense and anti-sense primers were 5′-CCCCAGTCACCTGCTGTTAT-3′ and 5′-TGGAATCCTGAACCCACTTC-3′, respectively. The expected size of the amplified fragment was 171 bp. For MCP-1 mRNA detection in decidual cell cultures the sense primer was 5′-GCTCAGCCAGATGCAA-3′ and the anti-sense primer was 5′-GTCCAGGTGGTCCATG-3′. The β-actin sense and anti-sense primers were 5′-CGTACCACTGGCATCGTGAT-3′ and 5′-GTGTTGGCGTACAGGTCTTTG-3′, respectively. The expected sizes of the amplified fragments for MCP-1 and β-actin mRNA were 452 and 459 bp, respectively. Comparisons of control and the various treatment groups were performed using the Kruskal-Wallis analysis of variance on ranks test followed by the Student-Newman-Keuls post hoc test with P value <0.05 representing statistical significance. For immunohistochemistry, the intensity of staining in control versus preeclampsia cases was compared using the χ2 test, with significance set at a probability value of <0.05. An increase in the numbers of CD68+ cells was observed in the decidua of preeclamptic mothers, compared to that of normal term pregnancies (Figure 1, A and B). Specifically, four control samples showed a complete absence of CD68+ cells in the decidua, two displayed sparsely isolated cells and only one specimen exhibiting numerous CD68+ cells. By contrast, each specimen of decidua from preeclamptic placentas displayed numerous immunoreactive cells. In three of the cases, the CD68+ cells were focally clustered. Statistical analysis showed that there was a difference in the distribution of case and control specimens such that a higher proportion of decidua from preeclamptic placentas were present in categories with a higher number of CD68+ cells (χ2 = 10.8; df = 3; P = 0.01). The localization of CD68+ cells in decidua was confirmed by staining serial sections for CD68 and the decidual cell marker vimentin (Figure 1, C and D). To demonstrate the expression of MCP-1 in vivo, RT-PCR analysis was performed on LCM-isolated decidual cells. As shown in Figure 2, a band corresponding in size to the MCP-1 mRNA product was obtained from the cDNA of the two specimens tested.Figure 2Reverse transcriptase-PCR analysis of MCP-1 mRNA levels in microdissected decidua tissues. Total RNA of two decidua specimens (lanes 1 and 3) was reverse-transcribed and amplified in the presence of MCP-1 primers. For each specimen a negative control lacking the reverse transcriptase was amplified and loaded onto the gel (lanes 2 and 4). Placental RNA was used as a positive control (PC). Forty-five cycles were run for each PCR. The size of the molecular weight makers (lane M; bp) is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because circulating levels of both E2 and progesterone rise during the first trimester, E2 was used as the control incubation for evaluating the effects of the progestin MPA. Figure 3 indicates that in cultures maintained in E2 alone, 10 ng/ml of TNF-α and IL-1β increased net immunoreactive MCP-1 outputs by 70.4-fold ± 16.4 and 149.6-fold ± 50.3, respectively (mean ± SEM, n = 7, P < 0.05). Specifically, incubation with TNF-α and IL-1β elevated MCP-1 output from 7.3 ± 2.4 pg/ml/μg protein in control cultures to 449.5 ± 174.6 and 631.3 ± 180.5 pg/ml/μg protein, respectively (P < 0.05). Similarly, in E2 plus MPA-treated cultures MCP-1 output was increased by 83.5-fold ± 20.6 and 103.1-fold ± 14.7, respectively with values increasing from basal levels of 6.1 ± 1.6 pg/ml/μg protein to 510.1 ± 172.4 and 576.0 ± 141.1 pg/ml/μg protein by
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