The PDE4 inhibitor rolipram prevents NF-kappaB binding activity and proinflammatory cytokine release in human chorionic cells

Abstract

Spontaneous preterm delivery is linked to intrauterine inflammation. Fetal membranes are involved in the inflammatory process as an important source of mediators, and the chorion leave produces high levels of the proinflammatory cytokine TNF-alpha when stimulated by LPS. The transcription factor NF-kappaB is the main regulator of this inflammatory process and controls the production of cytokines by the chorion leave. Phosphodiesterase 4 inhibitors are recognized for their anti-inflammatory and myorelaxant effects. The purpose of this study was to investigate whether PDE4 inhibition affects the LPS signaling in human cultured chorionic cells. We showed that these cells express TLR4, the main LPS receptor, and exhibit a predominant PDE4 activity. Upon LPS challenge, PDE4 activity increases concomitantly to the induction of the specific isoform PDE4B2 and chorionic cells secrete TNF-alpha. LPS induces the nuclear translocation of the NF-kappaB p65 subunit and the activation of three different NF-kappaB complexes in chorionic cells. The presence of the PDE4 inhibitor rolipram reduces the TNF-alpha production and the activation of the three NF-kappaB complexes. These data indicate that the PDE4 family interacts with the LPS signaling pathway during the inflammatory response of chorionic cells. PDE4 selective inhibitors may thus represent a new therapeutic approach in the management of inflammation-induced preterm delivery.

Figures

Fig 1. Characterization of cultured chorionic cells

Immunofluorescence analysis of CK7 (A), vimentin (B) and CD45 (C) in cultured chorionic cells showed the clustered pattern of trophoblasts, the expansion of mesencymal cells and a few macrophages.

Fig 2. Response of chorionic cells to LPS in the presence and in the absence of macrophages Concentration of TNFα and MCP1 were measured in cell media collected after 4h of treatment with LPS 100 ng/ml or its diluent. The cell preparations were either or not depleted of CD45 positive cells. Data are expressed as the mean ± sem of 5 different preparations. *, p

Fig 3. Analysis of TLR4 expression by chorionic cells

Lysates of cells treated with LPS 100 ng/mL for the indicated times were subjected to Western Blot (15 μg of proteins per lane) and probed with the TLR4 antibodies. Lysate of U937 cells served as a positive control. The detection of β-actin in each sample served as a loading control. A representative experiment is shown, reproduced 3 times.

Fig 4. Induction of PDE4 by LPS in chorionic cells

A. Time-dependent effect of LPS on PDE4 activity. Data reported the mean ± SEM of 4 separate experiments. **, p

Fig 5. Effect of LPS and rolipram on TNFα secretion by chorionic cells

A. Time-course of TNFα secretion by chorionic cells. Cells were incubated in the absence or the presence of LPS 100 ng/mL with or without rolipram 10−5 M for the indicated times. A representative experiment is shown, data reported the mean ± SD of triplicates in the assay, reproduced 3 times. B. Chorionic cells of 10 different placentas were incubated in the absence or presence of LPS with or without rolipram for 4h. Data are expressed as the mean ± SEM of the 10 separate experiments. *, p < 0.01, significantly different from control and rolipram. #, p < 0.01, significantly different from LPS treatment.

Fig 6. Effect of LPS and rolipram on the NF-κB p65 nuclear translocation in chorionic cells

A. p65 nuclear translocation assessed by immunofluorescence under LPS 100 ng/mL with or without rolipram 10−5 M for 2h. A representative experiment is shown, reproduced 7 times. B. Chorionic cells were incubated in the absence or in the presence of LPS and rolipram for 2h. p65 nuclear translocation was assessed by immunofluorescence and quantified as described in Materiel and Methods. Data are expressed as the mean ± SEM of the 7 separate experiments.*, p < 0.05, significantly different from control and rolipram. #, p < 0.01, significantly different from LPS treatment. C. Nuclear extracts from chorionic cells treated with LPS with or without rolipram for 2h were subjected to Western Blot (25 μg of proteins per lane) and probed with the p65 antibodies. The detection of β-actin in each sample served as a loading control. A representative experiment is shown, reproduced 3 times.

Fig 7

Effect of LPS and rolipram on the NF-κB binding activity in chorionic cells. Representative experiments are shown, reproduced 3 times. A. Time-course of NF-κB binding activity in chorionic cells under LPS 100 ng/mL with or without rolipram 10−5 M. Nuclear extracts from chorionic cells were incubated with biotin end-labeled oligonucleotides containing the NF-κB consensus sequence and subjected to EMSA. B. Supershift analysis of the NF-κB complexes present at 2h of LPS treatment. Nuclear extracts were pre-incubated with antibodies against p50 or p65.