Regulation of interleukin-6 expression in human decidual cells and its potential role in chorioamnionitis

Abstract

Chorioamnionitis frequently precedes both genital tract and placental inflammation and is both a primary cause of maternal morbidity and a major antecedent of preterm premature rupture of the membranes (PPROM) as well as preterm delivery (PTD). In most cases of chorioamnionitis, neutrophils dominate the decidua. In a subset of these cases, a predominance of monocytes is uniquely associated with both neonatal intraventricular hemorrhage and death. The multifunctional cytokine, interleukin-6, promotes local monocyte dominance via several mechanisms. In this study, immunostaining of placental sections revealed significantly higher interleukin-6 HSCOREs in decidual cells (DCs) but not in interstitial trophoblasts, in chorioamnionitis versus gestational age-matched control placentas (P < 0.05). In confluent leukocyte-free term DCs, secreted interleukin-6 levels in incubations with estradiol-17β were increased 2500-fold by IL-1β (P < 0.05). This up-regulation was inhibited by more than 50% in parallel incubations that included medroxyprogesterone acetate (n = 12, P < 0.05). Western blotting data confirmed these enzyme-linked immunosorbent assay results; quantitative RT-PCR findings demonstrated corresponding changes in interleukin-6 mRNA levels. Specific inhibitors of signaling for both nuclear factor-κB activation and p38-mitogen-activated protein kinase, but not for protein kinase C, significantly decreased IL-1β-enhanced interleukin-6 expression levels in cultured DCs. In conclusion, in situ and in vitro results indicate that significantly enhanced interleukin-6 expression levels in DCs during chorioamnionitis could be pivotal in skewing decidual monocyte differentiation to macrophages.

Figure 1

Immunohistochemical analysis of IL-6 expression in CAM-complicated decidua. CAM-complicated (A and B) and control (C and D) decidual specimens were immunostained to detect expressed IL-6 and vimentin in serial sections. Vimentin staining identified DCs (B and D). IL-6 immunoreactivity was more intense in decidual cells (arrows) of CAM-complicated specimens (A) than control specimens (C). Interstitial trophoblasts (arrowheads) displayed similar levels of IL-6 immunoreactivity in both specimen groups, but the localization of the staining was more perimembranous in CAM-complicated specimens, particularly when immediately adjacent to DCs (A, inset). The inset in A is a magnification of the outlined area. E: Staining with an isotype-matched, negative, control antibody. F: IL-6 immunoreactivity HSCOREs (mean ± SEM) in control (n = 9) and CAM-complicated (n = 10) specimens. *P < 0.05 in both CAM-complicated versus control DCs and DCs versus interstitial trophoblast in CAM-complicated specimens and Scale bars: 50 μm (A–F); 25 μm (inset).

Figure 2

Effects of MPA and IL-1β on IL-6 output by DC monolayers. Confluent, leukocyte-free term DCs were incubated for 7 days in 10⁻⁸ mol/L E₂ or E₂ + 10⁻⁷ mol/L MPA then switched to DM with corresponding steroid(s) ± 1 ng/ml IL-1β for 24 hours. IL-6 levels were measured by ELISA in conditioned DM and normalized to cell protein amount. Decidual specimens were obtained from separate patients (n = 12; mean ± SEM). The ordinate represents IL-6 (pg/ml/μg of cell protein) as linear plot (A) and log scale (B). *Versus E₂ or E₂ + MPA; **versus E₂ + IL-1β (P < 0.05).

Figure 3

Western blot of MPA and IL-1β effects on IL-6 output by DC monolayers. Confluent, leukocyte-free term DCs were incubated for 7 days in 10⁻⁸ mol/L E₂ or E₂ + 10⁻⁷ mol/L MPA, then switched to a DM with corresponding steroid(s) ± 1 ng/ml IL-1β for 24 hours. Conditioned DM was subjected to 10 to 20% SDS-polyacrylamide gel electrophoresis and Western blotting (see Materials and Methods). A representative blot at a 10-second exposure time is shown. Only the lanes relevant to this study are shown; the others have been omitted from the image.

Figure 4

Concentration-dependent effects of IL-1β on IL-6 output by DC monolayers maintained in E₂ + MPA. Confluent, leukocyte-free term trimester DCs were incubated for 7 days in 10⁻⁸ mol/L E₂ + 10⁻⁷ mol/L MPA and then were switched to a DM with steroids ± the indicated amount of IL-1β (0.01 to 10.0 ng/ml). IL-6 levels were measured by ELISA in conditioned DM and normalized to cell protein amount. Decidual specimens were obtained from separate patients (n = 5, mean ± SEM). *Versus E₂ + MPA; **versus 0.01 ng/ml IL-1β; ***versus 0.1 ng/ml IL-1β (P < 0.05).

Figure 5

Effects of MPA and IL-1β on IL-6 mRNA levels in DC monolayers. Confluent leukocyte-free term DCs were incubated for seven days in 10⁻⁸ mol/L E₂ or 10⁻⁸ mol/L E₂ + 10⁻⁷ mol/L MPA and then were switched to a DM with corresponding steroid(s) ± 1 ng/ml IL-1β for six hours. Aliquots of extracted total RNA were used to measure IL-6 mRNA levels by quantitative real-time RT-PCR. Decidual specimens were from separate patients (n = 8; mean ± SEM). The ordinate represents IL-6 mRNA/β-actin as a linear plot (A) and log scale (B). *Versus E₂ or E₂ + MPA; **versus E₂ + IL-1β (P < 0.05).

Figure 6

Effects of signaling pathway inhibitors on IL-6 secretion by term DC monolayers. Confluent, leukocyte-free term DCs were incubated for seven days in 10⁻⁸ mol/L E₂ + 10⁻⁷ mol/L MPA. Cultures were then switched for 24 hours to a DM with E₂ + MPA ± signaling pathway inhibitors ± 1 ng/ml IL-1β. The following specific inhibitors were used: p38 MAPK inhibitor (P38KI; SB203580), protein kinase C inhibitor (PKCI; calphostin C), and NF-κB activation inhibitor (NFkBI). All of the inhibitors were added as pretreatments for 30 to 60 minutes before the addition of IL-1β. Levels of IL-6 were measured by ELISA in conditioned DM and normalized to cell protein amount. Decidual specimens were obtained from separate patients (n = 6 each for E₂ + MPA and p38KI groups; n = 3 each for PCKI and NFkBI groups; mean ± SEM). The ordinate represents IL-6 (pg/ml/μg of cell protein). A: Effect of inhibitors on IL-1β-induced IL-6. B: Effect of inhibitors on IL-6 under basal conditions (E₂ + MPA). *Versus E₂ + MPA; **versus E₂ + MPA + IL-1β (P < 0.05). neg cont, negative control.

Figure 7

Western blot of effects of signaling pathway inhibitors on IL-6 secretion by term DC monolayers. Confluent, leukocyte-free term DCs were incubated for seven days in 10⁻⁸ mol/L E₂ + 10⁻⁷ mol/L MPA. Cultures were then switched for 24 hours to a DM with E₂ + MPA ± signaling pathway inhibitors ± 1 ng/ml IL-1β. The following specific inhibitors were used: p38 MAPK inhibitor (P38KI), protein kinase C inhibitor (PKCI), and NF-κB activation inhibitor (NFkBI). All of the inhibitors were added as pretreatments for 30 to 60 minutes before the addition of IL-1β. Conditioned DM was subjected to 12% SDS-polyacrylamide gel electrophoresis and Western blotting (see Materials and Methods). A representative blot at a 2-minute exposure time is shown. Positive control (Pos Cont): 10 ng of recombinant human IL-6.

Figure 8

Functional in vitro assay. IL-6 skews monocyte differentiation to macrophages. Monocytes from human peripheral blood were cultured for five days in a combination of conditioned medium obtained from term decidual cells that had been treated for 24 hours with E₂ or E₂ + IL-1β in the presence or absence of anti-IL-6 antibody (α-IL6) and 20 ng/ml M-CSF, 30 ng/ml GM-CSF, and 10 ng/ml IL-4. Differentiation was determined at day 5 by the expression of the macrophage markers CD14 (left) and CD68 (data not shown for CD68) and the dendritic cell marker CD11c (right). Data are shown as the mean ± SEM (n = 4). *P < 0.05; **P < 0.005.