Higher concentrations of inflammatory cytokines have been documented in amniotic fluid, cord blood and tracheal aspirates of premature infants who subsequently developed BPD (12
), whereas anti-inflammatory cytokines were found to be absent or in lower concentrations in bronchoalveolar lavage fluid of premature infants with BPD (17
). These patterns may depend upon the expression of pro- and anti-inflammatory cytokines in multiple placental compartments. The results of this study support the hypothesis that placental tissues of infants who developed BPD manifest down regulation of IL-10 expression, while robustly express IL-6.
Our original hypothesis proposed an imbalance of cytokines among infants who develop BPD with an increase in pro-inflammatory cytokines and a decrease in anti-inflammatory cytokines in the placenta. Postulating a predominance of placental IL-6 staining relative to IL-10 is based upon the observations that IL-6 is elevated in amniotic fluid, cord blood and tracheal aspirates of premature infants who subsequently develop BPD (12
). Furthermore, it is recognized that cytokines can be secreted from cells in the decidua and chorion, as well as from infiltrating macrophages (20
). One mechanism responsible for the activation of an inflammatory response in the premature lungs may be direct damage, as ventilator induced injury can induce mRNA expression of IL-6 in the preterm lung (22
). Inhalation of amniotic fluid by the fetus may favor the extension of the placental/amniotic fluid environment into the tracheobronchial tree, thus promoting inflammation, as proposed by Ghezzi et al, (14
). Alternatively, placental expression of pro-inflammatory mediators may prompt a fetal systemic inflammatory response with increased endothelial permeability, capillary leakage and diffuse alveolar damage predisposing to BPD. Increased cord blood concentration of IL-6 among infants destined to develop BPD is supportive of this notion (12
). The results of this study, in contrast, did not demonstrate differences in IL-6 staining in any of the placental compartments of infants with BPD compared to those without BPD. Similar expression of IL-6 in placental tissue with differences in IL-10 expression would certainly not preclude more prominent IL-6 production in other tissues and body fluid in response to inflammation and lung injury. We propose that in the absence of placental IL-10 in BPD cases, the inflammatory function of IL-6 remains unchecked.
It is interesting that we did not see a difference in the incidence of histological chorioamnionitis between newborns with and without BPD. Whereas some studies have found a positive association between chorioamnionitis and BPD (8
), other studies have not supported the notion of chorioamnionitis as a risk factor for BPD (23
). This conflicting data likely represents differences in definitions of chorioamnionitis and BPD, time periods studied, and antenatal factors, such as maternal steroid use. Chorioamnionitis is characterized by higher amniotic fluid and cord blood concentrations of IL-6 (25
); however, no studies specifically examined the relationship between chorioamnionitis, as defined by neutrophil response, and placental cytokine expression. The relationship between the BPD pathology and cytokine response is likely more complex than we understand.
The results of this study support a critical anti-inflammatory and immunomodulatory role of IL-10 even before birth in the development of BPD. Recent reports have suggested that IL-10 placental expression is gestational age dependent (27
) and we did not see prominent IL-10 staining in the cord, membranes, and placenta. Yet, in spite of this, the presence of placental IL-10 in the villous trophoblast was associated with a reduction in the odds of developing BPD. This relationship was present in unadjusted and adjusted analyses for differences in gestational age and race. In agreement with our findings, is the observation that the absence of IL-10 in tracheal aspirates in infants <27 weeks is associated with a higher risk of developing BPD (16
). Similarly, poor IL-10 expression has been reported in preterm infants with hyaline membrane disease (18
). While these latter studies did not specifically look at chronic lung disease, it may be that persistent and unchecked inflammation plays a role in the evolution of BPD.
Recent work has shown genetic heritability to BPD (8
). Emerging data suggests that genetic variations in IL-10 gene regulation may ultimately affect the anti-inflammatory response. IL-10 regulation is complex and it is subject to genetic influence (29
). The single-nucleotide polymorphism (SNP) at −1082 G allele has been associated with a higher IL-10 expression in certain cell types (31
). Moreover, recent work has shown that preterm infants who carry two G alleles of the IL-10 (−1082) SNP were at a prominently reduced risk for neonatal cerebral, eye, and lung (specifically BPD) damage (32
). Although it is plausible that polymorphic changes in the IL-10 promoter may be differentially utilized in different cell types in response to inflammatory triggers, our data suggest that IL-10 expression in the villous tissue is not uniquely influenced by chorioamnionitis. We propose that IL-10 down-regulation in the placenta of infants with BPD involves additional events, such as an excessive production of prostanoids or dysregulation of the hormonal cascade.
There are important limitations to this study that should be acknowledged. Although we matched for variables (sex, period of birth, and birthweight) that previously have shown to affect BPD (33
), there may be unidentified variable influencing the results of a case controlled retrospective study. While we felt matching for the above variables was clinically relevant, we acknowledge potential bias in case selection in a retrospective analysis. The cohort of this study represents approximately 46% and 23% of BPD and no BPD infants, respectively, available from our NICU. Despite our matching of the above variables, there were group differences in gestational age and trends in differences in race. Racial differences in the development of BPD have been previously reported and matching on race would have made it impossible to attain a reasonable sample size (35
). The lower gestational age and race were adjusted for in the logistic regressions. We also are aware that there are other criteria being used by some to define BPD, such as an oxygen challenge reduction test (36
). This methodology was not being performed at the time period of our study and our definition of oxygen use at 36 weeks PMA is within the scope of the National Institute of Health consensus conference definition of BPD (37
We also recognize the variability of immunohistochemical staining and the subjectivity of grading. To allow for a more accurate statistical analysis, each batch of staining consisted of patients with BPD and their matched controls, in addition to a standard reference control. The scoring system used for determining the extent of staining was qualitative rather than quantitative; ideally a system that determines extent of cytokine staining both in terms of placental area involved and degree of staining would be desirable. In spite of this, the analysis of inter-observer reliability was reassuring with a kappa value of 0.82. In addition, the categorical analysis of our data as either “no staining” or “any staining” also decreased subjectivity.
Our findings support a “cytokine balance” theory. Logistic regression analysis shows an independent association between IL-10 and BPD. This suggests that a baseline inflammation is present, but the absence of anti-inflammatory mediators in the placenta may initiate fetal inflammatory responses. We feel that this study is important as it presents a prenatal or antenatal environment that may ultimately affect the postnatal course of an infant. Further studies of placental examination may allow us to better identify those premature infants at increased risk for particular morbidities.