Progestin-inflammatory cytokine interactions affect matrix metalloproteinase-1 and -3 expression in term decidual cells: implications for treatment of chorioamnionitis-induced preterm delivery

Abstract

Context: Chorioamnionitis (CAM)-elicited preterm delivery (PTD) is associated with elevated amniotic fluid levels of IL-1beta and TNF-alpha. We hypothesized that IL-1beta and TNF-alpha may induce matrix metalloproteinase (MMP)-1 and MMP-3 activity to promote PTD by degrading decidual and fetal membranes and cervical extracellular matrix.

Objective: Our objective was to evaluate: 1) MMP-1 and MMP-3 expression in decidual sections from uncomplicated term, idiopathic preterm, and CAM-complicated deliveries, and 2) the separate and interactive effects of IL-1beta, TNF-alpha, medroxyprogesterone acetate (MPA), and a p38 MAPK inhibitor (SB203580) on MMP-1 and MMP-3 expression in term decidual cells (DCs).

Interventions and main outcome measures: Decidua were immunostained for MMP-1 and MMP-3. Cultured term DCs were incubated with estradiol (E2) or E2 plus MPA with or without IL-1beta or TNF-alpha with or without SB203580. ELISA and Western blotting assessed secreted MMP-1 and MMP-3 levels, quantitative real-time RT-PCR assessed mRNA levels, and substrate gel zymography was used to determined MMP-1 and MMP-3 proteolytic activity.

Results: MMP-1 and MMP-3 immunostaining was more prominent in CAM-complicated decidua vs. control preterm and term decidual specimens (P < 0.05). Compared with basal outputs by DCs incubated with E2, TNF-alpha enhanced MMP-1 and MMP-3 secretion by 14 +/- 3- and 9 +/- 2-fold, respectively, and IL-1beta increased MMP-1 and MMP-3 secretion by 13 +/- 3- and 19 +/- 2-fold, respectively (P < 0.05). Addition of MPA lowered basal MMP-1 and MMP-3 outputs by 70%, whereas the TNF-alpha- and IL-1beta-enhanced MMP-1 and MMP-3 levels were blunted by more than 50% (P < 0.05). SB203580 suppressed TNF-alpha- and IL-1beta-induced MMP-1 and MMP-3 secretion by severalfold. Western blotting confirmed the ELISA results, and mRNA levels corresponded with MMP-1 and MMP-3 protein levels. MMP-1 and MMP-3 proteolytic activity was confirmed by substrate gel zymography.

Conclusion: Augmented DC-expressed MMP-1 and MMP-3 in CAM-complicated pregnancies may promote PTD via decidual, fetal membrane, and cervical extracellular matrix degradation. Effects of progestin-p38 MAPK signaling inhibition on cytokine-enhanced MMP-1 and MMP-3 expression in term DCs suggest alternative mechanisms to prevent CAM-induced PTD.

Figure 1

Immunoreactive MMP-1 and MMP-3 expression in decidual cells in placental sections from term, preterm, and CAM-complicated deliveries. Representative photomicrographs of immunohistochemical staining for MMP-1 and MMP-3 in term decidua (A and D, respectively), preterm decidua (B and E, respectively), and CAM-complicated decidua (C and F, respectively) with positive signals are in red. Decidual cell-enriched areas (D) and trophoblast-enriched areas (T) are noted. Vimentin staining (I) was used to identify decidual cells. Staining with isotype-matched control antibody (G) revealed no positive staining. HSCORE analysis (H) revealed significantly higher levels of MMP-1 and MMP-3 staining in CAM-complicated tissues (n = 10), compared with tissues from term (n = 10) and preterm (n = 7) deliveries (*, P < 0.001 vs. term and preterm). Magnifications, ×200.

Figure 2

Concentration-dependent effects of TNF-α and IL-1β on MMP-1 and MMP-3 output by E2- plus MPA-treated term decidual cell monolayers. Confluent, leukocyte-free decidual cells were incubated for 7 d in 10⁻⁸ m E2 plus 10⁻⁷ m MPA and then switched to DM with steroids alone or together with TNF-α (A) or IL-1β (B) for 24 h. The abscissa indicates cytokine concentrations in nanograms per milliliter. Levels of MMP-1 and MMP-3 were measured by ELISAs in conditioned DM and normalized to cell protein (mean ± sem; n = 3). Note the different values on the ordinates corresponding to MMP-1 and MMP-3. *, P < 0.05 vs. E2 plus MPA.

Figure 3

Effects of E2, MPA, TNF-α, and IL-1β on MMP-1 and MMP-3 output by term decidual cell monolayers. Confluent, leukocyte-free decidual cells were incubated for 7 d in 10⁻⁸ m E2 or E2 plus 10⁻⁷ m MPA and then switched to DM with corresponding steroid(s) with or without 1 ng/ml TNF-α or IL-1β for 24 h. Levels of MMP-1 and MMP-3 were measured by ELISA in conditioned DM and normalized to cell protein (mean ± sem). P < 0.05: *, vs. E2; **, vs. E2 plus MPA; †, vs. corresponding fold change for E2 plus TNF-α; ‡, vs. corresponding fold change for E2 plus IL-1β (mean ± sem). A, MMP-1 (n = 7); B, MMP-3 (n = 8); C, MMP-1 and MMP-3 (n = 7 or 8).

Figure 4

A, Western blot analysis of E2, MPA, TNF-α, IL-1β, and p38 MAPK inhibitor SB effects on MMP-1 and MMP-3 output by term decidual cell monolayers. Confluent, leukocyte-free decidual cells were incubated for 7 d in 10⁻⁸ m E2 or E2 plus 10⁻⁷ m MPA and then switched to DM with corresponding steroid(s) in the presence or absence of 1 ng/ml TNF-α or IL-1β for 24 h with or without exposure to 10⁻⁵ m SB 30 min before treatment. Aliquots of conditioned DM were subjected to Western blot analysis. Results of a representative experiment are shown. B, Substrate gel zymography of E2, MPA, TNF-α, and IL-1β effects on MMP activity by term decidual cell monolayers. Confluent, leukocyte-free decidual cells were primed for 7 d in DM containing vehicle control (C), 10⁻⁸ m E2, or E2 plus 10⁻⁷ m MPA and then incubated for 24 h in DM with corresponding steroid(s) in the presence or absence of 1 ng/ml TNF-α or IL-1β. Conditioned DM was run on a 12% casein gel zymogram and then subjected to renaturing and staining procedures. Proteolytic activity of the active MMPs can be seen as clear bands around 45 kDa in the darkly stained gel (representative zymogram shown).

Figure 5

ELISA analysis of E2, MPA, TNF-α, and IL-1β, and p38 MAPK inhibitor SB effects on MMP-1 and MMP-3 output by term decidual cell monolayers. Confluent, leukocyte-free decidual cells were incubated for 7 d in 10⁻⁸ m E2 plus 10⁻⁷ m MPA and then switched to DM with corresponding steroids in the presence or absence of 1 ng/ml TNF-α or IL-1β for 24 h with or without exposure to 10⁻⁵ m SB 30 min before treatment. MMP-1 and MMP-3 levels were quantified by ELISA in conditioned DM and normalized to cell protein (mean ± sd; n = 2).

Figure 6

Effects of E2, MPA, TNF-α, and IL-1β on MMP-1 and MMP-3 mRNA levels in term decidual cell monolayers. Confluent, leukocyte-free decidual cells were incubated for 7 d in 10⁻⁸ m E2 plus 10⁻⁷ m MPA and then switched to DM with E2 plus 10⁻⁷ m MPA alone or together with TNF-α or IL-1β for 6 h. Quantitative real-time RT-PCR was performed on extracted RNA for MMP-1 and MMP-3 and normalized to β-actin mRNA (mean ± sem, n = 4). P < 0.05: *, vs. E2; **, vs. E2 plus MPA; †, vs. corresponding E2 plus TNF-α; ‡, vs. corresponding E2 plus IL-1β. A, MMP-1 mRNA/β-actin mRNA; B, MMP-3 mRNA/β-actin mRNA.