Our study demonstrates that the risk of BPD among infants born before the 28th week of gestation is associated with elevated blood concentrations of a variety of proteins integral to inflammation. These include pro-inflammatory cytokines, adhesion molecules and proteases. Reduced risk is prominently associated with increased concentrations of one chemokine, RANTES. These findings suggest that an inflammatory process involving a variety of mediators is critical in the development of BPD. Elevations of inflammatory proteins associated with BPD risk occur during the first days following birth. However, inflammation intensifies thereafter. Therefore, exposures that promote inflammation after the first postnatal days may be more critical in the development of BPD.
Our observations are in general agreement with previous reports. For example, in a cohort of ELGANs, death or BPD at 36 weeks PMA was predicted by elevated blood concentrations of a variety of cytokines (7
). The notable similarities to our findings were the increased risk associated with high levels of TNFα and decreased risk with high levels of RANTES. In contrast to that study, we did not find a distinct pattern of decreased concentrations of proteins that the authors associated with adaptive (as opposed to innate) immunity in infants who developed BPD, with the exception of RANTES. Our choice of proteins might have masked these trends. For example, we did not measure IFNγ or TNFβ, cytokines associated with innate immunity, or IL-10, a cytokine associated with adaptive immunity (14
). A more important difference between these studies might be our exclusion of infants who died prior to 36 weeks PMA. In some studies of BPD, death prior to 36 weeks PMA is included as an outcome measure because it competes with BPD. However, because a relatively small proportion of infants die from lung disease, and some die of multi-system organ failure or sepsis, processes with a strong systemic inflammatory component, we believe that it is more appropriate to exclude infants who die prior to 36 weeks PMA when investigating the role of inflammation in lung disease.
We observed a doubling of risk of less severe BPD among infants with concentrations of TNFα in the highest quartile on the first postnatal day. A similar association was not seen in another cohort of premature infants (15
). Several important differences in study design might explain this lack of agreement. For example, that study included infants born before the 33rd
week of gestation, while our study did not include any infant born between weeks 28 to 32. The differences may also have resulted from different methods to account for the potential contribution of chorioamnionitis. We did not adjust for the presence of chorioamnionitis in our regression models because in a previous study of BPD risk in this cohort, we did not identify chorioamnionitis as a risk factor (16
). In addition, that study measured concentrations in cord blood only, while the specimens in our study were obtained postnatally. In both humans and animals, pro-inflammatory cytokines appear in the lungs soon after early neonatal exposures (e.g. mechanical ventilation) that promote inflammation (17
), raising the possibility that the early elevations in TNFα in our infants are a consequence of postnatal exposures.
The chemokine RANTES was one of the few proteins associated with reduced risk of both mild/moderate and severe BPD. As a chemotactic agent it attracts inflammatory cells to the site of infection or injury (19
). Therefore, one might expect elevated levels to intensify the inflammatory process and increase the likelihood of BPD. In fact, elevated levels of other chemotactic agents, including IL-8 and MIP-1, have been observed in bronchial alveolar lavage fluid in infants who develop BPD (20
). However, there is evidence that RANTES actually protects against organ damage in animal models of inflammation-mediated diseases (21
). In addition, an in vitro
study provides support that RANTES reduces inflammation (23
). Thus the association of reduced risk with elevated concentrations of RANTES might reflect anti-inflammation and protection.
Other proteins highly associated with both mild/moderate and severe BPD are adhesion molecules, most notably ICAM-1 and ICAM-3. As a group, adhesion molecules promote migration of inflammatory cells from the blood to sites of injury in the lung, and their primary site of action is at the blood-tissue interface (24
). Because of this site of action, blood levels of adhesion molecules might be elevated in organ-specific inflammation. Therefore, it is not surprising that increased concentrations in blood are also associated with BPD risk. Other circulating inflammatory proteins that are elevated in BPD may not be of pulmonary origin. Rather, their presence may result from a systemic response to injury or inflammation in the lung (e.g., SAA or CRP). It is also possible that under certain circumstances proteins produced by cells in the lung remain in the lung compartment, while under other circumstances (e.g., intense pulmonary inflammation), they may “leak” into the circulation (25
). Because of these uncertainties, we advise caution in drawing inferences about the observed changes in blood proteins and pulmonary pathology.
Some important observations resulted from developing time-oriented risk models for BPD that both included and excluded specific, known clinical risk factors. In one model, we included birth weight Z-score, a marker of fetal growth restriction, because it is both a known BPD risk factor (16
) and is associated with decreased placental expression of selected inflammatory cytokines (27
). The associations between protein concentrations and BPD risk in this model were similar to those in the model that excluded birth weight Z-score. This finding suggests that the increased BPD risk associated with fetal growth restriction is probably not mediated by circulating inflammation-associated proteins. By contrast, the addition of mechanical ventilation at 7 days changed the apparent influence of ICAM-1 and RANTES, suggesting that mechanical ventilation may influence pathogenesis by altering expression of these proteins.
This study has a number of strengths. We included a large number of infants, making it unlikely that we missed important associations due to lack of statistical power, and we collected all of our data prospectively. We selected infants based on GA, not birth weight, in order to minimize confounding due to factors related to fetal growth restriction, and with regression models we adjusted for fetal growth restriction. We did not include infants who died prior to 36 weeks PMA, thereby restricting this to a study of BPD and not the composite outcome of BPD or death.
This study also has limitations. For practical reasons, our cohort was limited to infants who had complete neurodevelopmental evaluation at 24 months. This resulted in the non-random exclusion of two potentially important cohorts. The incidence of both mild/moderate and severe BPD in the cohort of infants who survived to 24 months but for whom protein measurements were not available was only slightly lower than in our study cohort. Therefore, their exclusion is not likely to have resulted in a significant bias in the results. The incidence of severe BPD among infants who died between 36 weeks PMA and 24 months was significantly higher (47%) than in our study cohort (9%). The effect of exclusion of these infants, however, is likely to be minimal because they represent a small proportion of the ELGAN Study cohort (≈ 4%). The diagnosis of BPD was made on the basis of clinicians’ decisions to treat with mechanical ventilation or supplemental oxygen, not on the basis of physiologic disturbances, and clinical practices almost certainly varied among centers. Finally, because this was an observational study, we cannot know whether the protein elevations were associated with BPD in a cause and effect relationship or whether the elevations are surrogates for other processes that might have influenced BPD risk.
Our study has several implications for researchers interested in preventing BPD. First, the different risk factor profile for mild/moderate and severe BPD suggest that BPD is a heterogenous condition. Consideration of this heterogeneity could lead to more informative epidemiologic studies. Second, our findings suggest the possibility that inflammation-associated proteins in neonatal blood could serve as biomarkers of modifiable biological processes involved in the development of BPD. If so, these biomarkers could be used as response measures in intervention studies. Third, at least some aspect of the fetal and neonatal inflammatory response is associated with a lower risk of BPD. Thus one approach to prevention might be the enhancement of endogenous protectors.