Bronchopulmonary dysplasia (BPD) is the most common, serious sequela of premature birth. Inflammation is a major contributor to the pathogenesis of BPD. Often initiated by a pulmonary fetal inflammatory response, lung inflammation is exacerbated by mechanical ventilation and exposure to supplemental oxygen. In response to these initiators of injury, a complex interaction occurs between proteins that attract inflammatory cells (ie, chemokines), proteins that facilitate the transendothelial migration of inflammatory cells from blood vessels (ie, adhesion molecules), proteins that promote tissue damage (ie, pro-inflammatory cytokines and proteases), and proteins that modulate the process (eg, anti-inflammatory cytokines, binding proteins and receptor antagonists). In addition, during recovery from inflammatory injury, growth factors and other substances that control normal lung growth and mediate repair influence subsequent lung structure. In this review, we discuss the role of each aspect of the inflammatory process in the development of BPD. This discussion will include data from measurements of biomarkers in samples of fluid aspirated from the airways of human infants relevant to each phase of inflammation. Despite their limitations, these measurements provide some insight into the role of inflammation in the development of BPD and may be useful in identifying infants at risk for the disease.
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Improvement in the care of premature infants in the past several decades has resulted in increased survival of extremely premature infants.1 However, significant morbidity among these infants still occurs, the most common serious morbidity being bronchopulmonary dysplasia (BPD). Contrary to expectations, surfactant treatment and newer modes of mechanical ventilation have not reduced the gestational-age-specific incidence of BPD. Therefore, the prevalence of BPD has actually increased in recent decades. When first described, BPD was characterised histologically by tissue destruction and fibrosis, and clinically by severe impairment of oxygenation and ventilation. In recent years, a less severe disease has emerged. This “new” BPD is characterised by large terminal air sacs and incomplete alveolarisation but little fibrosis,2 and manifests clinically as mild impairment of lung function.
Lung inflammation is a major contributor to the pathogenesis of BPD. Often initiated by a pulmonary fetal inflammatory response to bacteria and other organisms in the amniotic cavity, the lung inflammation is exacerbated by mechanical ventilation and exposure to supplemental oxygen. In response to these initiators of lung injury, a complex interaction occurs between proteins that attract cells integral to the inflammatory process (ie, chemokines), proteins that facilitate the transendothelial migration of inflammatory cells from blood vessels (ie, adhesion molecules), proteins that promote tissue damage (ie, pro-inflammatory cytokines and proteases), and proteins that modulate the process (eg, anti-inflammatory cytokines, binding proteins and receptor antagonists). In addition, hormones, growth factors and other substances that control lung cell homoeostasis may influence recovery from inflammatory injury (fig 1).
UTILITY OF BIOMARKERS OF LUNG INFLAMMATION
A considerable body of literature provides valuable insight into the role of inflammation in BPD by describing inflammatory processes after lung injury in animal models. These studies offer advantages over studies performed in human neonates. For example, experimental conditions can be tightly controlled to isolate the influence of discrete elements of the inflammatory cascade. Cell-bound mediators, or those confined to intracellular spaces, can be examined because of the availability of lung tissue. Unfortunately, results of studies from animal models are not easily extrapolated to human neonates. They are not conducted in the context often encountered in human premature birth (eg, chorioamnionitis). They usually investigate the response to a single initiator of injury (eg, oxidative stress). Most investigations have been acute phase studies only and have not investigated the time-oriented changes in inflammatory mediators.
Studies of inflammatory mediators in human neonates avoid some of these problems but also have limitations. Lung tissue is not available except from infants who have succumbed to the disease, and fluid from the airways is only available from intubated infants. Therefore, measurements in healthy infants to serve as controls are usually lacking. Measurement of analytes in lung epithelial lining fluid (ELF) is limited to mediators that are present in this fluid; intracellular proteins (eg, intracellular transcription factors) cannot be analysed in this manner. In addition, the quantity of mediators in ELF may not reflect biological activity because of varying degrees of tissue binding. In the past, the number of proteins that could be measured in a single specimen was limited because of the requirement of single analyte assay techniques such as ELISA. Mulitplexed assays are now available that permit the simultaneous measurement of a large number of biomarkers in a small volume of biological fluid.
An alternative to the measurement of biomarkers in ELF is to measure changes in genes that are responsible for the production of specific proteins in the inflammatory process. Individual gene expression can be determined by measuring the mRNA of candidate proteins. Multiple genes can be examined in this manner using highly automated gene chip array assays. Unfortunately, this powerful tool requires a source of RNA, and in premature neonates, the sole source from the lung is cells collected during tracheal aspiration (TA). Therefore, the proteins that can be examined are limited almost exclusively to those that are expressed in airway inflammatory cells.
Samples of ELF can be collected by one of two techniques: TA after instillation of saline into the trachea or by small volume bronchoalveolar lavage (BAL).3 4 Both techniques result in recovery of sufficient quantities of protein to permit measurements of inflammatory proteins and growth factors. Biomarkers in BAL appear to reflect a more distal origin compared with TA, and, because of the probable site of inflammation during lung injury, would be preferable. However, BAL often causes bleeding into the fluid (in ∼50% of samples), which can perturb the measurement of many biomarkers and is not a component of the routine care of premature infants. Therefore, nearly all studies of biomarkers in human infants use tracheal aspirate collected during routine suctioning as the source of biological material. In contrast with BAL, the volume recovered during tracheal suctioning is variable. Therefore, quantities of analytes in TA specimens must be expressed using an internal standard other than volume. The content of urea nitrogen, the secretory component of IgA (sIgA) and total protein5 have all been used as internal standards. There does not appear to be an influx of either urea nitrogen or sIgA in the presence of epithelial disruption, in contrast with total protein, making total protein potentially less useful in the presence of severe lung inflammation. Urea nitrogen may be preferable relative to sIgA in the presence of lung infection.
Despite the limitations, quantities of substances in the airways of neonates that are integral to the inflammatory process can provide information about the milieu of the lung and underlying disease states. This information is valuable because it may assist in the understanding of the pathophysiology of inflammation-mediated lung diseases. It may also identify modifiable risk factors for these diseases and may permit the early identification of infants who might best benefit from prophylactic or therapeutic modalities (eg, anti-inflammatory agents).
NEONATAL LUNG INFLAMMATION AND ASSOCIATED BIOMARKERS
In this review, we discuss briefly the role of each aspect of the inflammatory process in the development of BPD. This discussion includes data from measurements of biomarkers in ELF from human infants relevant to each phase of inflammation (table 1). Selected references to studies in animals and adult humans are included only when necessary to clarify an aspect of the role of a mediator. A comprehensive review of the literature is not included; previously published articles provide additional information.6–9
Lung inflammation in the neonate may be initiated by events that occur before birth (eg, exposure to intrauterine infection) or as a result of respiratory therapies used to treat lung disease (ie, supplemental oxygen and mechanical ventilation). After the initiation of the inflammatory process, a cascade of events occurs beginning with the recruitment of inflammatory cells to the lung,6 10 a process that it is mediated by proteins called chemokines. A role for chemokines in the development of BPD is suggested by the presence of increased quantities of a number of chemokines in ELF from infants who develop BPD. The predominant cell type that appears first is the neutrophil, followed by an influx of macrophages and other cells.
Chemokines are categorised into four subtypes (CC, CSC, CX3C and XC), of which three contain proteins that are associated with inflammatory lung diseases (CC, CSC and CX3C).11 Interleukin (IL) 8 (also called CXCL8) is the chemokine that has been investigated most extensively in preterm infants.5 12–18 Increased concentrations of IL8 precede neutrophil infiltration in tracheal aspirates from preterm infants19 as well as in infants ventilated with high tidal volumes.20 In infants with BPD, concentrations of IL8 in tracheal aspirates are raised during the first 10 days of life compared with those in infants who do not develop BPD.21 In multivariable analysis, high concentrations of IL8 in tracheal aspirates collected at birth are associated with prolonged duration of mechanical ventilation in infants less than 28 weeks’ gestation.12 Although these observations suggest that an inflammatory process that results in later persistent lung injury begins with the production of chemokines, raised IL8 concentrations should be interpreted with caution. For example, in one study of ventilated preterm infants, concentrations of IL8 in tracheal aspirates predicted the combined outcome of death or BPD but not BPD alone,14 suggesting that IL8 concentrations may be a marker for general illness severity but are not specific for BPD.
Other chemokines associated with the development of BPD include the monocyte chemoattractant proteins, MCP-1 (CCL2), MCP-2 (CCL8) and MCP-3 (CCL7), and macrophage inflammatory proteins, MIP-1α (CCL3) and MIP-1β (CCL4).22 In addition to an association with BPD, increased concentrations of MCP-1 in airway secretions have been observed in the presence of tracheal colonisation of Ureaplasma urealyticum, a putative risk factor for BPD.16 Other forms of lung injury, for example pulmonary haemorrhage, also appear to result in increased concentrations of the chemokines, MCP-1, MCP-2 and MCP-3.22 Other proteins with chemotactic activity associated with the influx of cellular elements after inflammatory lung injury include leukotriene B4 and the anaphylatoxin C5a.23 24
A critical step in the recruitment of inflammatory cells is transmigration of these cells from capillaries to air spaces or the extracellular matrix of the interstitium. The initial step in this process is a phenomenon known as rolling, during which inflammatory cells migrate to the endothelial surface and move slowly along the surface. This is followed by transient, then firm, adhesion and finally transmigration from the capillary. Adhesion molecules are proteins located on the cell surface and are essential to these processes. These proteins appear to be critical in the development of parenchymal damage in infants with BPD. Adhesion molecules include selectins—responsible for transient adhesion and rolling—and integrins and immunoglobulins—responsible for firm adhesion and transmigration.25
Intercellular adhesion molecule-1 (ICAM-1) is a member of the IgM super-family which promotes adhesion. Increased airway concentrations of soluble ICAM-1 have been observed at 10 days of age in infants who develop BPD.21 Concentrations of soluble L-selectin in tracheal aspirates from infants who do and do not develop BPD are similar on the first day of life, but are raised at day 7 of life, in infants who develop BPD compared with both healthy infants and infants with respiratory disease syndrome who do not develop BPD.25
The tissue damage associated with inflammatory injury is mediated by pro-inflammatory cytokines. These cytokines are synthesised by neutrophils and other inflammatory cells in the airway and interstitium, in addition to a variety of cells of the lung parenchyma (eg, airway and alveolar epithelial cells, endothelium and fibroblasts). Cytokines bind to specific cell-surface receptors, and act as mediators in an immune response via intracellular signalling which includes the upregulation and/or downregulation of genes and their transcription factors. For example, the activation of a nuclear factor-κB pathway appears to be a critical mechanism in lung injury in premature infants based on its upregulation after mechanical ventilation.26
The common pro-inflammatory cytokines tumour necrosis factor alpha (TNFα), IL1, IL6 and IL8 are the most extensively studied cytokines in this category, and are important biomarkers for the prediction of adverse pulmonary outcomes in preterm infants.27–29 Increased concentrations of IL1β in tracheal aspirates predict the requirement for mechanical ventilation and oxygen supplementation.29 30 Increased concentrations of IL1, TNFα, IL6 and IL8 correlate with the duration of supplemental oxygen and mechanical ventilation and are increased in infants who develop BPD compared with infants of similar gestational age who do not develop BPD.27 Increased IL1β concentrations and IL1β/IL6 ratios are also associated with risk factors for BPD, specifically colonisation with Ureaplasma urealyticum.28 TNFα, which appears during the early phases of an inflammatory response, is raised in BAL samples from ventilated preterm infants with poor pulmonary outcomes.31
Proteases and their inhibitors
Matrix metalloproteases (MMPs), formerly known as collagenases, are a family of proteinases that are critical in the damage resulting from inflammatory lung injury because they cause destruction of the alveolar/capillary interface and extracellular matrix proteins. They also play an important role in normal lung development.32 Because they may be both beneficial and destructive, their activity is regulated closely by a balance between factors that regulate their production and activation, and the production of specific proteinase inhibitors. Preterm infants appear to have an imbalance between the proteinase and proteinase inhibitor system, which may potentiate lung damage.33 High concentrations of MMPs (particularly MMP8 and MMP9) and/or low concentrations of tissue inhibitor of metalloproteases in TA fluid are associated with the development of BPD.34 35 An imbalance between cysteine proteases and their inhibitors,36 and disturbances in the production and destruction of elastin may also be critical in the pathogenesis of BPD.37 38 The increase in pulmonary trypsin in tracheal aspirates of infants developing BPD suggests a role for this potent matrix-degrading proteinase and MMP activator.39
Reactive oxygen species (ROS) and oxidative injury
Cell damage induced by exposure to high concentrations of inspired oxygen results in part from the production by neutrophils of ROS. ROS cause tissue damage by lipid peroxidation.40 A common site of damage is the basement membrane and other elements of the lung matrix. One consequence of this damage is increased microvascular permeability and vascular leakage, resulting in oedema formation,23 a potential contributor to the development of BPD. In addition, ROS potentiate tissue damage by inhibiting antiproteases that modulate the activity of proteases such as elastase. Biomarkers of oxidative stress include increased myeloperoxidase activity and quantities of xanthine oxidase. Concentrations of these markers are raised during the first week of life in tracheal aspirates from infants who develop BPD compared with concentrations in infants who are ventilated but recover without BPD.41 These raised concentrations are associated with increased elastase activity, perhaps as a result of inhibition of its respective antiprotease. Premature infants may be particularly vulnerable to this effect of ROS because they have a relative deficiency of these antiproteases by virtue of their prematurity. In addition, preterm infants appear to have deficient quantities of enzymes responsible for scavenging ROS, including superoxide dismutase and glutathione peroxidase.42 43
Hyperoxic lung injury may have important secondary effects. For example, hyperoxia may stimulate excessive production of bombesine-like peptides (BLPs).44 BLPs are growth factors known for their stimulatory effect on fetal lung development. However, raised urinary BLP concentrations are associated with an increased risk for the development of BPD, presumably because they also function as pro-inflammatory cytokines.45 46
After exposure to oxygen and mechanical ventilation, most infants recover without developing BPD. In these infants, the anti-inflammatory cytokines appear to play a dominant role. A number of studies support the hypothesis that premature infants are particularly vulnerable because they underexpress damage protectors, anti-inflammatory cytokines and other proteins that localise or downregulate the inflammatory process. The anti-inflammatory cytokine IL10 has been the subject of investigation in premature infants. IL10 inhibits the production of TNFα, IL1β, IL6 and IL8, and upregulates the receptor antagonist for IL1, and by these mechanisms may protect the lung.10 47 Although preterm infants have an acute phase inflammatory response that is similar to the response in term infants, during the first 4 days of life, IL10 is measurable in airway secretions of term infants but usually is not present in the secretions of preterm infants.48 This may be due to a relative inability of the lung macrophage in the preterm compared with the term infant to produce IL10.49 IL10 concentrations in the BAL fluid from infants developing BPD rise earlier, but fall faster, than concentrations in infants who do not develop BPD, and increased quantities of IL10 are observed in tracheal aspirates from infants with acute lung disease who recover without BPD.50 51
Another anti-inflammatory cytokine that has been hypothesised to have a role in the development of BPD is the Clara cell protein (CC10). CC10 is produced predominantly by the Clara cell, a pulmonary mucosal epithelial cell. CC10 inhibits inflammatory cell chemotaxis and fibronectin binding. In addition, it may be critical in protecting the surfactant system during lung inflammation because it inhibits the secretion of phospholipase A2, which degrades the phospholipid component of surfactant. Low concentrations of CC10 are found in tracheal aspirates from premature infants compared with concentrations in adults,52 and an inverse relationship between CC10 concentration and risk of BPD is observed.53 These findings suggest that CC10 may be developmentally regulated, and the relative deficiency seen in premature infants may increase the risk of lung injury.
Lung development is highly programmed and regulated by a variety of growth factors and hormones.54 Lung injury and associated inflammation can perturb the production of these proteins and alter lung growth and development after injury. For example, the production of transforming growth factor (TGF) β may be upregulated after lung injury, providing a protective effect by reducing pro-inflammatory cytokine production.55 However, its overexpression may result in the stimulation of fibroblasts and lung fibrosis, one of the hallmarks of severe BPD. This overexpression of TGFβ can be identified by increased concentrations in airway secretions and may be a predictive marker for severe of BPD.56 Overexpression of TGFα, a growth factor critical to normal lung development in transgenic mice, results in the development of fewer and larger alveoli,57 histological features of BPD that predominate in milder forms of the disease, in addition to lung fibrosis.
A number of other growth factors play important roles in fetal lung development and therefore are presumably critical in normal lung development in prematurely born infants. These include neuregulin,58 epithelial growth factor,59 insulin-like growth factor60 and fibroblast growth factors,61 which also appear to protect alveoli from damage.62 Unfortunately, the role of these growth factors in the development of BPD remains unclear.
There has been much recent interest in the role of angiogenic growth factors, specifically vascular endothelial growth factor (VEGF), in BPD. VEGF, which controls the growth of blood vessels, is expressed in the endothelium and epithelium in the developing lung, and plays a critical role in alveolar development because of the close association between angiogenesis and lung development.63 Concentrations of VEGF are decreased in the tracheal aspirate of infants developing BPD.64 The arrest in alveolar development, accompanied by a paucity of pulmonary capillaries seen in animal models of mild BPD, may be the result of downregulation of VEGF expression.
GENETIC PREDISPOSITION TO INFLAMMATION
Individual infants may be peculiarly vulnerable to BPD because of predisposing genetic factors. For example, male gender appears to increase the likelihood of severe acute lung disease and BPD.65 The manner by which gender and other genetic factors influence lung disease is not entirely understood. One possibility is genetically determined variability in the level of protein production (eg, increased pro-inflammatory cytokines or decreased anti-inflammatory cytokines) in response to initiators of inflammation. Certain genetic risk factors, including polymorphisms in pro-inflammatory cytokines, have been identified in other pulmonary diseases (eg, asthma).66
Although the link between genetic risk and infantile lung disease appears to be likely, proof of altered genes as a contributing cause is limited to date to mutations that affect the metabolism of surfactant proteins. These mutations cause disease primarily in mature neonates, or later in life. In preterm infants, a number of investigators have examined the relationship between candidate gene single nucleotide polymorphisms (SNPs) and BPD. These studies have examined the association between BPD and SNPs for the gene expression for TNFα (specifically −308 SNP67–70 and the −1031, −863, −857 and −238 SNPs), IL1β, its receptor antagonist, TGFβ1 and MCP-1. Data from these studies suggest that these SNPs do not play a significant role in determining risk for BPD, although one study reported that the adenine allele of TNFα −238 may reduce the incidence of BPD.71 An explanation for the lack of success of these studies is the likelihood that BPD occurs only when a genetically vulnerable person is exposed to the necessary initiators and promoters of inflammation. Therefore, identifying SNPs that impart risk will require interpretation in the context of these exposures. Although the role of genetic biomarkers in defining risk of BPD remains uncertain, future medical care may include identifying vulnerable people by testing for critical gene markers of susceptibility.
UNRESOLVED QUESTIONS AND FUTURE RESEARCH
Although studies of biomarkers in human neonates have provided some insight into the role of inflammation in the development of BPD, most previous studies have limitations. Few have searched rigorously for evidence of early initiators of inflammation, for example chorioamnionitis, funisitis or other factors that might influence vulnerability of the lung to inflammatory injury. Many studies have examined mediators at a single time point, a method that ignores the phasic nature of the inflammatory process. Those that have followed inflammatory mediators over time have investigated an insufficient number of mediators to draw reasonable conclusions about the interactive effects of damage promoters and protectors. Most studies lack context specificity. The concept of context specificity is critical in evaluating the role of inflammatory mediators. Under one set of circumstances, some cytokines contribute to programmed cell death, yet under other circumstances they reduce the probability of cell death.72 Relationships between damage promoters, modulators and protectors change rapidly. For example, TNFα, a pro-inflammatory cytokine, can be a strong stimulus for the production of IL10, an anti-inflammatory cytokine, which in turn can downregulate expression of TNFα.73 Only by repeatedly measuring many relevant initiators, promoters, modulators and protectors at multiple time points can the role of inflammation in the development of BPD be adequately investigated.
Future investigation of the role of lung inflammation in the development of BPD should include large time-oriented epidemiological studies that thoroughly identify all aspects of the perinatal milieu. These studies will then be capable of incorporating the concepts of context specificity in creating risk profiles that characterise each infant’s vulnerability. Risk profiles will permit us to group infants who are truly comparable, and thus will have the potential to improve the quality of future observational studies and clinical trials. Such studies may facilitate laboratory investigation of molecular biological mechanisms of injury and the development of targeted therapies.
CLB received salary support from the Thrasher Research Fund. CELD received salary support from the Susan B Saltonstall Foundation and the National Institutes of Health (HL 37930).
Competing interests: None.