We examined the association between elevated concentrations of 25 blood proteins in the first two postnatal weeks and the risk of EPPD and PD among extremely preterm infants. In light of evidence that pulmonary inflammation contributes to the occurrence of CLD (3
) we hypothesized that systemic inflammation would be present in infants who developed EPPD, and to lesser degree in infants who developed PD.
Only a small amount of predictive information about PD and EPPD is available in blood spots collected on day 1, with increasing information on day 7, and even more on day 14. The number of inflammation-related proteins associated with EPPD increased with increasing postnatal age raising the possibility that systemic inflammation associated with lung dysfunction reflects postnatal phenomena. This is no surprise since infants who experience EPPD have such inflammation-promoting exposures as supplemental oxygen and mechanical ventilation, which are viewed as antecedents of CLD (21
Elevations of inflammation-associated proteins provided more information about reduced risk of PD rather than increased risk. No protein concentration elevation on any of the three days was associated with increased risk of PD. In contrast, the elevated concentrations of three proteins on day 7 (RANTES, VCAM-1 and MMP-1) and two on day 14 (RANTES and VEGF) were associated with reduced risk. On one level, this is evidence that inflammation does not contribute substantially to the occurrence of PD. Our findings raise the possibility that inflammation protects. We are reluctant to accept this view just yet. Rather, we think our provocative findings need to be replicated and alternative explanations need to be considered.
RANTES has been described as pleiotropic (23
). Some of the functions of this protein contribute to reducing inflammation (24
). Consequently, our finding elevated concentrations of RANTES associated with reduced risk of EPPD and PD might be viewed as additional support for a modulation of pre-existing inflammation not otherwise apparent to us. Low concentrations of RANTES in blood shortly after birth have also been associated with increased risk of BPD or death (3
). Elevated concentrations of VEGF were associated with reduced risk of PD. Support for the hypothesis that low VEGF levels contribute to PD comes from the observation that VEGF preserves alveolar development, and that reduced concentrations of lung VEGF disrupt lung growth and vascular distributions within the lung (26
As with PD, elevations of some inflammation-associated proteins, including RANTES and VEGF, are associated with a decreased risk of EPPD. However, in contrast to PD, elevations of some proteins are associated with an increased risk of EPPD. On day 14, elevated concentrations of two proteins (IL-8 and ICAM-1) were associated with increased risk of EPPD. The association with IL-8 is not surprising because of its pro-inflammatory properties. ICAM-1 is an adhesion molecule that mediates the adhesion of white blood cells to the vascular endothelium and is important for recruiting leukocytes to sites of inflammation. Higher concentrations of soluble ICAM-1 have been associated with poor outcomes in adults with acute lung injury (28
Our findings are best interpreted in light of the current understanding of the biology of inflammation. Individual proteins are only a small part of a much larger process. Inflammation-related proteins are highly interrelated(29
), and together appear to contribute to organ damage (30
). As a result, each protein is best interpreted as representing the interlinked web of proteins indicating inflammation, and possibly promoting lung damage or phenomena likely to reduce damage. An inflammatory stimulus alters the expression of more than a thousand genes, increasing the expression of hundreds of proteins, and decreasing the expression of hundreds of others(31
). For example, when the expression of VCAM-1 is increased, so, too, is the expression of other markers of inflammation (32
). Consequently, what we see is a very limited snapshot of what has preceded and/or accompanied the development of lung dysfunction.
Lung dysfunction can promote and/or exacerbate an inflammatory response. Hypercapnia promotes adhesion molecule expression in both inflammatory-stimulated human pulmonary microvascular endothelial cells and in an animal model of inflammatory-induced acute lung injury (33
). This, and similar observations, suggest that some of the protein concentration changes we found might be a consequence, rather than an antecedent of lung dysfunction. Such a response might very well further contribute to lung dysfunction, as lung inflammation can promote more lung inflammation. Lung exposed to an inflammatory stimulus releases circulating mediators that induce liver vascular endothelial cells to activate NF-κB and to express ICAM-1 and VCAM-1(34
). In essence, a self-reinforcing loop is generated that promotes sustained inflammation, thereby increasing the risk of inflammation-induced damage.
We do not know to what extent protein concentrations in the blood are of pulmonary origin. It is possible that under certain circumstances proteins produced by cells in the lung remain in the lung compartment, while under other circumstances (e.g., intense or prolonged pulmonary inflammation), they enter the circulation. It is possible that infants with PD and EPPD have inflammatory processes that are more prominent in the lung than in the blood. That is, systemic inflammation is not a prominent component of early pulmonary dysfunction and our examination of proteins in the blood may provide only a limited and biased view of the inflammatory processes associated with early pulmonary dysfunction. Analysis of inflammatory proteins from the epithelial lung fluid might help elucidate this possibility.
Our study has several strengths and limitations. We created time-oriented risk models to provide information about the relative significance of protein concentrations at critical postnatal ages. We included a large number of infants, making it unlikely that we have missed important associations due to lack of statistical power. We selected infants based on GA, not birth weight, in order to minimize confounding due to factors related to fetal growth restriction (35
The weaknesses of our study are those of all observational studies. We do not know to what extent the protein reductions or elevations contributed to PD or EPPD and to what extent the protein reductions or elevations are surrogates for other processes that might have influenced CLD risk. We did not include infants who died prior to 24 months, and more infants died in the EPPD group and PD group than in the Low FiO2 group. This might bias our results if infants died because of a systemic inflammatory process. In addition, we collected all of our data prospectively. Eluting materiel from blood spots is likely to include the intracellular contents of disrupted cells in the circulation. Thus, what we measured is probably the sum of proteins in the circulation, and proteins released from cells whose integrity was lost in the drying of the blood spots.
In conclusion, elevated concentrations of inflammation-associated proteins in the blood were associated with reduced risk of PD. A similar pattern was seen to a lesser extent among children who had EPPD, although elevated concentrations of other inflammation-related proteins were associated with increased risk. The lack of evidence of inflammation in the blood early in the neonatal period among children who had EPPD or PD suggests that systemic inflammation is not a process that begins in utero. Postnatal events or exposures are more likely to be the promoters of this phenomenon. Because of the strong association between EPPD and CLD, and to a lesser degree PD and CLD, targeted therapies designed to modulate specific elements of systemic inflammation may modify CLD risk.