Bronchoalveolar lavage fluid from preterm infants with chorioamnionitis inhibits alveolar epithelial repair

Abstract

Background: Preterm infants are highly susceptible to lung injury. While both chorioamnionitis and antenatal steroids induce lung maturation, chorioamnionitis is also associated with adverse lung development. We investigated the ability of bronchoalveolar lavage fluid (BALF) from ventilated preterm infants to restore alveolar epithelial integrity after injury in vitro, depending on whether or not they were exposed to chorioamnionitis or antenatal steroids. For this purpose, a translational model for alveolar epithelial repair was developed and characterised.

Methods: BALF was added to mechanically wounded monolayers of A549 cells. Wound closure was quantified over time and compared between preterm infants (gestational age < 32 wks) exposed or not exposed to chorioamnionitis and antenatal steroids (>or= 1 dose). Furthermore, keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF) were quantified in BALF, and their ability to induce alveolar epithelial repair was evaluated in the model.

Results: On day 0/1, BALF from infants exposed to antenatal steroids significantly increased epithelial repair (40.3 +/- 35.5 vs. -6.3 +/- 75.0% above control/mg protein), while chorioamnionitis decreased wound-healing capacity of BALF (-2.9 +/- 87.1 vs. 40.2 +/- 36.9% above control/mg protein). BALF from patients with chorioamnionitis contained less KGF (11 (0-27) vs. 0 (0-4) pg/ml) and less detectable VEGF (66 vs. 95%) on day 0. BALF levels of VEGF and KGF correlated with its ability to induce wound repair. Moreover, KGF stimulated epithelial repair dose-dependently, although the low levels in BALF suggest KGF is not a major modulator of BALF-induced wound repair. VEGF also stimulated alveolar epithelial repair, an effect that was blocked by addition of soluble VEGF receptor-1 (sVEGFr1/Flt-1). However, BALF-induced wound repair was not significantly affected by addition of sVEGFr1.

Conclusion: Antenatal steroids improve the ability of BALF derived from preterm infants to stimulate alveolar epithelial repair in vitro. Conversely, chorioamnionitis is associated with decreased wound-healing capacity of BALF. A definite role for KGF and VEGF in either process could not be established. Decreased ability to induce alveolar epithelial repair after injury may contribute to the association between chorioamnionitis and adverse lung development in mechanically ventilated preterm infants.

Figure 1

Characterisation of the alveolar epithelial wound repair model. A. Effect of addition of various concentrations of fetal bovine serum (FBS) to culture medium on in vitro alveolar epithelial wound healing (A; t = 24 hours). Bars represent mean wound closure + SEM (number of pixels ×1000) of triplicate experiments (*p < 0.05; ***p < 0.001 vs. control). B. Differences in wound closure rate over time between incubation in control medium (0.1% FBS) and positive control medium (10% FBS). Points represent mean wound closure rate + SEM of triplicate experiments (**p < 0.01; ***p < 0.001 vs control). C. Effect of addition of various concentrations of bronchoalveolar lavage fluid (BALF) from a term newborn diluted in control medium (0.1% FBS) on in vitro alveolar epithelial wound healing (t = 24 hours). Bars represent mean wound closure + SEM (% of control) of triplicate experiments (*p < 0.05; **p < 0.01 vs. control). D. Effects of various concentrations of albumin and BALF (diluted 1:10) from a term newborn (total protein in the original BALF specimen was 1.7 mg/ml). Bars represent mean wound closure + SEM relative to control medium of triplicate experiments (***p < 0.001 vs. control).

Figure 2

Effects of chorioamnionitis and antenatal steroids on bronchoalveolar lavage fluid (BALF) wound healing capacity. Effect of BALF obtained at different postnatal days from patients with and without chorioamnionitis (A), and from patients with and without antenatal steroid exposure (B) on in vitro alveolar epithelial wound healing. Bars represent mean wound closure +/- SEM (% above control/mg total protein in BALF) of triplicate experiments (*p < 0.05).

Figure 3

M30 detected in culture supernatant after day 0/1 wound healing experiment according to chorioamnionitis or betamethasone exposure. Bars represent mean wound closure + SEM per group. No significant differences between groups were present.

Figure 4

Keratinocyte growth factor (KGF) and vascular endothelial growth factor (VEGF) levels in bronchoalveolar lavage fluid (BALF). KGF (A) and VEGF (B) levels in bronchoalveolar lavage fluid (BALF) obtained at different postnatal days from patients with and without chorioamnionitis. Points represent individual BALF growth factor concentrations, horizontal lines represent medians (*p < 0.05).

Figure 5

Vascular endothelial growth factor (VEGF) and keratinocyte growth factor (KGF) effects on alveolar epithelial wound repair. Effect of addition of different combinations of bronchoalveolar lavage fluid (BALF), VEGF (10 pg/ml), and soluble VEGF-receptor 1 (sVEGFr1; 6 ng/ml) (A), or various concentrations of KGF (B) on in vitro alveolar epithelial wound healing. Bars represent mean wound closure + SEM (% of control) of triplicate experiments (*p < 0.05, and **p < 0.01 vs control). No significant differences are present between BALF alone and BALF with any addition. Significant differences between VEGF, and both sVEGFr1 and VEGF + sVEGFr1 are present (both p < .05).