Mechanisms of abruption-induced premature rupture of the fetal membranes: thrombin-enhanced interleukin-8 expression in term decidua

Abstract

Recent evidence has linked preterm premature rupture of the fetal membranes (PPROM) to placental abruption. Because neutrophils are a rich source of proteases that can degrade extracellular matrix in abruption-associated PPROM, we examined whether decidual neutrophil infiltration complicates abruption-associated PPROM. Accordingly, immunostaining for the neutrophil marker CD15 was performed in placentas obtained after overt abruption (decidual hemorrhage) with or without PPROM and in control placentas. Abruptions were associated with a marked decidual neutrophil infiltration that peaked after PPROM, whereas decidua from gestational age-matched controls were virtually devoid of neutrophils. Neutrophil infiltrates co-localized with fibrin deposition. Because abruptions elicit intense decidua-enhanced thrombin production, we examined the regulation of abruption-induced neutrophil infiltration. Expression of the primary neutrophil chemoattractant interleukin-8 (IL-8) was evaluated in leukocyte-free term decidual cells incubated with estradiol (E2; control) or with E2+medroxyprogesterone acetate (to mimic pregnancy)+/-thrombin. After 24 hours, enzyme-linked immunosorbent assay measurements indicated that thrombin (0.1 to 2.5 U/ml) elicited a dose-dependent elevation in secreted IL-8 (P<0.05) with 2.5 U/ml of thrombin increasing IL-8 levels by >14-fold in E2 and E2+medroxyprogesterone incubations. Results were validated by Western blot and quantitative reverse transcriptase-polymerase chain reaction. In summary, thrombin-enhanced IL-8 expression in term decidual cells may explain how abruption-associated PPROM promotes decidual neutrophil infiltration.

Figure 1

Immunolocalization of CD15-positive cells (brown stain; details in Materials and Methods) in term (A) and preterm (B) control specimens as well as in patients with abruption (C and D). E: Conspicuous deposition of fibrin stained in red by the Picro-Mallory reagent. Decidua cells (arrow) are visible under the fibrin clot. F: CD15 immunostaining on a consecutive section. Note numerous CD15-positive cells in the decidua and in the fibrin clot. Original magnifications: ×200 (A–D); ×40 (E, F).

Figure 2

Effects of E₂, MPA, and thrombin on IL-8 output by DC monolayers. Confluent passaged, 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) ± 2.5 U/ml thrombin (Th) for 24 hours. IL-8 levels were measured by ELISA in conditioned DM and normalized to cell protein (details in Materials and Methods). *P < 0.05, E₂ versus E₂ + Th; **P < 0.05 E₂ + MPA versus E₂ + MPA + Th (n = 10 for all groups, mean ± SEM).

Figure 3

Dose response effects of thrombin on IL-8 output by DC monolayers maintained in the presence of E₂ + MPA. Confluent DCs were incubated for 7 days in 10⁻⁸ mol/L E₂ + 10⁻⁷ mol/L MPA, then switched to DM with corresponding steroid(s) ± 0.1 U/ml (low), 0.5 U/ml (med), and 2.5 U/ml (hi) thrombin (Thr) for 24 hours. IL-8 levels were measured by ELISA in conditioned DM and normalized to cell protein (details in Materials and Methods). *Versus E₂ + MPA; **versus E₂ + MPA + Th (low); and ***versus E₂ + MPA + Th (med) (P < 0.05, mean ± SEM, and n = 10 for all groups).

Figure 4

Western blot of thrombin effects on IL-8 output by in vitro DCs. Confluent 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) ± 2.5 U/ml thrombin (Th) for 24 hours. The conditioned medium was then subjected to Western blotting (see Materials and Methods). Lane 1: E2; lane 2: E2 + Th; lane 3: E2 + MPA; and lane 4: E2 + MPA + Th.

Figure 5

Effects of hirudin, thrombin, or both on IL-8 output by DC monolayers maintained in the presence of E₂ + MPA. Confluent DCs were incubated for 7 days in 10⁻⁸ mol/L E₂ + 10⁻⁷ mol/L MPA, then switched to DM with corresponding steroid(s) ± 0.5 U/ml thrombin (Th), or 0.5 or 2 U/ml hirudin (Hir), or hirudin plus thrombin for 24 hours. IL-8 levels in conditioned DM were measured by ELISA and normalized to cell protein (mean ± SD, n = 2) (details in Materials and Methods).

Figure 6

Quantitative RT-PCR of effects of E_2, MPA, and thrombin on IL-8 mRNA levels in leukocyte-free term DC monolayers. Confluent 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) ± 2.5 U/ml thrombin (Thr) for 5 hours. IL-8 mRNA levels were measured by quantitative RT-PCR of thrombin in conditioned DM and normalized to β-actin mRNA levels (details in Materials and Methods). Ordinate: IL-8 mRNA/β actin mRNA. *Versus E_2, **versus E₂ + MPA (P < 0.05, mean ± SEM, and n = 4 for all groups).