Maternal alcohol ingestion reduces surfactant protein A expression by preterm fetal lung epithelia

Abstract

In addition to neurodevelopmental effects, alcohol consumption at high levels during pregnancy is associated with immunomodulation and premature birth. Premature birth, in turn, is associated with increased susceptibility to various infectious agents such as respiratory syncytial virus (RSV). The initial line of pulmonary innate defense includes the mucociliary apparatus, which expels microorganisms trapped within the airway secretions. Surfactant proteins A and D (SP-A and SP-D, respectively) are additional components of pulmonary innate immunity and have an important role in pulmonary defense against inhaled pathogens. The purpose of this study was to determine if chronic alcohol consumption during the third trimester of pregnancy alters the function of the mucociliary apparatus and expression of SP-A and SP-D of fetal lung epithelia. Sixteen, date-mated ewes were assigned to two different groups; an ethanol-exposed group in which ewes received ethanol through surgically implanted intra-abomasal cannula during the third trimester of pregnancy, and a control group in which ewes received the equivalent amount of water instead of ethanol. Within these two groups, ewes were further randomly assigned to a full-term group in which the lambs were naturally delivered, and a preterm group in which the lambs were delivered prematurely via an abdominal incision and uterotomy. Ethanol was administered five times a week as a 40% solution at 1g/kg of body weight. The mean maternal serum alcohol concentration measured 6h postadministration was 16.3+/-4.36 mg/dl. Tracheas from six full-term lambs were collected to assess ciliary beat frequency (CBF). The lung tissue from all (24) lambs was collected for immunohistochemistry analysis of SP-A and SP-D protein production and fluorogenic real-time quantitative polymerase chain reaction analysis of SP-A and SP-D mRNA levels. Exposure to ethanol during pregnancy significantly blocked stimulated increase in CBF through ethanol-mediated desensitization of cAMP-dependent protein kinase. In addition, preterm born/ethanol-exposed lambs showed significantly decreased SP-A mRNA expression when compared with the preterm born/control group (P=.004); no significant changes were seen with SP-D. The full-term/ethanol-exposed lambs had no significant alterations in mRNA levels, but had significantly less detectable SP-A protein when compared with the full-term/control lambs (P=.02). These findings suggest that chronic maternal ethanol consumption during the third trimester of pregnancy alters innate immune gene expression in fetal lung. These alterations may underlie increased susceptibility of preterm infants, exposed to ethanol in utero, to RSV and other microbial agents.

Figure 1

Intra-abomasal cannula for alcohol delivery. The canula is present along the ventral right paralumbar area. To deliver alcohol, the screw-cap is released and a catheter is inserted and alcohol enters by gravity flow.

Figure 2

Ewes were exposed to alcohol as described. 6 full-term lambs were sacrificed and tracheal ring cilia were incubated with control media or 100 μM isoproterenol (ISO) for 30 min. CBF (panel A) and PKA (panel B) were assayed. ISO significantly stimulates increased lamb tracheal epithelial CBF and PKA from control ewes (*p<0.05; n=3 lambs). Maternal alcohol exposure desensitizes the lamb cilia to beta-agonist stimulated increases in CBF and PKA (n=3 lambs).

Figure 3

The relative SP-A mRNA expression in pre-term control (PTC), and full-term control (FTC) lambs as well as pre-term alcohol-exposed (in utero) (PTA) and full-term alcohol-exposed (in utero) lambs (FTA). The FTC and PTC values are equal to one. The PTA and FTA values are presented relative to the corresponding controls. The SP-A mRNA expression is significantly reduced (p=0.004) in PTA lambs (n=6 lambs) when compared to PTC lambs (n=6lambs). There is no significant change in SP-A mRNA expression between FTA and FTC lambs.

Figure 4

The relative SP-D mRNA expression in pre-term and full-term lambs with or without (control) maternal alcohol administration during gestation. Although maternal alcohol administration had a tendency to decrease the SP-D mRNA expression in the pre-term/alcohol exposed lambs (PTS), (n=6 lambs) when compared to the pre-term/control lambs (PTC), (n=6 lambs), this difference was not statistically significant (p=0.4). There is no significant change in SP-D mRNA expression between full-term/alcohol exposed and full-term/control lambs.

Figure 5

Representative immunohistochemical detection of SP-A protein in bronchial/bronchiolar epithelium of the full-term/alcohol exposed lambs (A), full-term control lambs (B), pre-term/alcohol exposed lambs (C) and pre-term control lambs (D). High distribution and intensity of staining is seen in pre-term lambs receiving alcohol, suggesting that alcohol may affect SP-A protein release from epithelial cells. Arrows depict epithelial cells with SP-A protein expression.