PERSPECTIVE
Understanding the Mechanisms of Infant Respiratory Distress and Chronic Lung Disease

Ian B. Copland and Martin Post

Department of Lung Biology and Pediatrics, The Hospital for Sick Children; and The University of Toronto, Toronto, Ontario, Canada

Respiratory distress syndrome in newborn infants (IRDS) is the most frequent respiratory cause of death and morbidity in children < 1 yr of age (1). Therapy for IRDS has been increasingly effective in reducing mortality at the expense of an increasing number of preterm survivors with chronic lung disease. This is particularly evident in extremely low birth weight (0.5-1.0 kg) infants, born between 24 and 28 wk (2). The lungs of these infants are structurally immature, often surfactant-deficient, fluid filled, and not supported by a stiff chest wall (3), which enhances their susceptibility to lung injury. Recent pathology of chronic lung disease (CLD) differs from that described previously by Northway and colleagues (4) in that the lungs are more uniformly inflated and have minimal airway injury, but demonstrate alveolar hypoplasia (5). CLD is a heterogeneous condition, with a variety of spectra; however, a cardinal event, which appears to be common to all types of CLD, is an inflammatory response. Inflammation can interfere with normal anatomic development of the airways and alveoli, and can lead to lung tissue damage. This tissue damage is exacerbated by the fact that the healing process is generally abnormal in the premature infant due to immaturity. Consequently, excessive inflammation and abnormal healing can result in dysplasia and metaplasia of the respiratory system.

	Genetic Risk of Developing IRDS

Until recently, respiratory distress has most commonly been attributed to developmental immaturity; however, the genetic risk for respiratory distress in infancy has been increasingly recognized. Reports of family clusters of affected infants and of ethnic- and gender-based respiratory phenotypes point to the contribution of inheritance. Similarly, different outcomes among gestation-matched infants with comparable exposures to oxygen, mechanical ventilation, or nutritional deficiency also suggest a genetic risk for respiratory distress (6). Genetic variations in surfactant protein (SP) genes A and B have provided the first examples of genetic risk of developing respiratory distress. Although ablation of the SP-A in mice does not result in respiratory distress for prematurely born pups (7, 8), differences in inheritance of SP-A alleles 1 and 2 in a Finish population has been shown to associate with different frequencies of respiratory distress (9). Conversely, infants homozygous for a mutation in codon 121 in exon 4 of SP-B consistently develop respiratory distress within the first 12-24 h of life (10, 11) and genetic disruption of SP-B in mice causes obvious neonatal respiratory distress (12). Thus, it appears that different SP-A gene alleles may be indirectly linked to IRDS, whereas loss of function of SP-B is clearly associated with IRDS. Aside from the surfactant protein genes, disruptions of several extrapulmonary genes have also been shown to produce neonatal respiratory distress. For example, disrupting the biologic function of GlcNAc N-deacetylase/N-sulfotransferase-1 (NDST-1) by homologous gene recombination in mice results in mice that develop respiratory distress (13). The respiratory distress is characterized by atelectasis due to type II pneumocyte immaturity, and a reduction of total phospholipids and disaturated phosphatidylcholine content. Respiratory distress is also found in mice lacking tumor necrosis factor (TNF)--converting enzyme, which is responsible for the proteolytic shedding of membrane-bound cytokines and growth factor precursors into soluble intercellular ligands (14), and in mice missing the Hoxa-5 gene, which affects the pulmonary epithelial expression of Nkx2.1 (TTF-1), Foxa2 (HNF-3), and N-myc (15). Mice lacking the -subunit of EnaC display respiratory distress because of improper lung liquid clearance (16). Other transgenes showing respiratory distress include mice lacking the glucocorticoid receptor (17) or cytochrome P450 1A2 (18). Hopefully, further identification of genetic markers for IRDS will lead to the development of treatment strategies for genetic lung disorders of infancy and assist in more accurate counseling of families whose infants are at genetic risk for development of respiratory distress.

	Cytokine Networks in IRDS and CLD

Current evidence unequivocally shows that lung inflammation is involved in the pathogenesis of IRDS and CLD, and identifying the cytokines which are associated with IRDS and CLD inflammation has been the focus of numerous studies. Together, these studies have demonstrated that infants with CLD have significantly higher soluble interleukin (IL)-2 receptor (a marker of lymphocyte activation) levels in their plasma than either RDS or control infants (19) and sometimes have increased bronchoalveolar lavage (BAL) or plasma levels of proinflammatory cytokines such as IL-1, IL-6, IL-8, and TNF- (20, 21). Interestingly, preterm newborns lack the ability to express anti-inflammatory cytokines such as IL-10, which may predispose premature infants to chronic lung inflammation (22).

Although numerous cytokines have been identified to associate with IRDS and CLD, the underlying pathogenic properties of these cytokines are only partially understood. Cytokines, whether they are called interleukins, colony-stimulating factors, interferons, or peptide growth factors, tend to act in networks or cascades, and within these networks a cytokine can be multifunctional, can demonstrate redundancy, synergistic activity, and often can antagonize other cytokine functions (23). Understanding the components of a cytokine network within a particular tissue and how these cytokines interact must be delineated if we are to understand how cytokines mediate the pathogenesis of IRDS and CLD. In this issue, Price and coworkers (24) show that different cytokines vary in their ability to regulate the expression of binding proteins for insulin-like growth factors at both the level of transcription and degradation. In particular, IL-1 and TNF-, which are associated with the pathogenesis of IRDS and CLD, can increase the levels of insulin-like growth factor (IGF)BP-3 and IGFBP-4 in a dose-dependent manner. There is ample evidence to suggest that the IGF system has a role in lung development (25) and that the presence of the IGF-binding proteins can determine the response of a cell to IGFs (26). Transcripts for IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 have been detected in the developing lung (26), and although their functions are unknown, the temporal and spatial gene expression patterns of IGFBPs (27, 28) suggest distinct roles for these proteins in controlling IGF action during lung development. With regard to IRDS and CLD, studies have found changes in IGFBP expression in a variety of lung injury models. Hyperoxia exposure of A549 cells induces growth arrest and increases IGFBP-2 and IGFBP-3 expression (29). These increases in IGFBPs are likely controlled via nuclear factor (NF)-B (30). Clinically it has been found that children with interstitial lung disease have increased amounts of IGFBP-2 in their BAL (31). Together, these studies demonstrate that the IGF-binding proteins could be important mediators in the cytokine network of the lung, and suggest that these proteins may play an important role in the injury-repair process, which is often impaired in premature infants at risk of IRDS and CLD.

Aside from delineating the components of the cytokine networks in IRDS and CLD, it is vital that we also isolate the upstream and downstream regulators, which initiate and amplify the cytokine networks within the premature lung (see Figure 1). An understanding of the expression of genes, in particular those genes that regulate the expression of cytokines, is critical in isolating key components involved in the evolution of CLD. NF-B represents one such upstream mediator, as it enhances the expression of many cytokine genes involved in inflammation (i.e., TNF-, IL-6, IL-8, and ICAM-1) (32). Other potential candidate mediators include activator proteins (AP) 1 and 2, the glucocorticoid receptor, cAMP response element binding proteins (CREB) CCAAT/enhancer binding proteins (c/EBP), Octamer factors, and Ets factors (33). Once the cytokine genes have been transcribed and translated, they can bind to their receptors, which can activate numerous signal transduction pathways. Pathways activated by cytokine/ receptor binding include the JAK/STAT pathway, the Ras-Raf-MAP kinase pathway, as well as the PI3-kinase pathway (34). The ability to activate multiple and overlapping signaling pathways explains why cytokines can be multifunctional and why they often show redundancy in function with other cytokines. If we are truly to understand the cytokine network involved in the pathogenesis of IRDS and CLD, cell culture and animal models must mimic the environment of the premature lung and be sophisticated enough to deal with the multiple signals that can induce the expression of cytokines and multiple signaling pathways cytokines can use to exert their biologic function. Up to now, studies investigating cytokines in IRDS and CLD have largely been descriptive, but recent advances in cell culture and genetics have provided researchers the tools to systematically elucidate the underlying mechanisms of the cytokine networks in IRDS and CLD.

View larger version (34K): [in this window] [in a new window]   Figure 1.   Schematic representation of the multiple factors that can stimulate the expression of a specific cytokine, the variety of effects once its mRNA has been translated into a protein, and the multiple signaling pathways which can evoke the biologic functions of that cytokine.

	Future Directions

Although some studies can be performed in cultured cells, tissues, and organs, findings from in vitro models must ultimately be tested on intact animals. Thus, animals are the best model system to understand the mechanisms of IRDS and CLD. The appropriateness of a specific animal model, however, is dependent on several factors. Specifically, how well does species, age, lung development, and response to injury reflect what occurs in humans? Can you measure the desired outcomes in your animal model? Does the agent you are studying have the same biologic effects, pharmacology, and toxicology in the animal model as in humans? Presently, the best model to study IRDS and CLD is the premature baboon model. Coalson and coworkers (38) have demonstrated that extremely premature baboons, despite appropriate oxygenation and ventilatory strategies, exhibit alveolar hypoplasia, saccular wall fibrosis, diminished capillary vasculature, and significant elevations in TNF-, IL-6, and IL-8 levels in tracheal aspirate fluids. This model mimics the human condition, but is problematic due to the high costs as well as the long duration of experiments and the fact that most primates are threatened species. Rodents, specifically the transgenic mouse, have become an increasingly popular alternative model for studying human diseases. Rodents are born with a saccular lung that is comparable to the human lung at 24-26 wk of gestation (39), and therefore are a promising experimental model to study IRDS and CLD in extremely low birth weight infants (2). Transgenic mice that overexpress the proinflammatory cytokine TNF- display reduced alveolarization (40) and could represent a model in which clinical treatment for CLD could be tested.

With sufficiently complex models, we can dissect the interactions between individual genes within cytokine networks and determine the effect of interventions and environmental factors. Specifically, we can determine how factors such as oxygen or mechanical ventilation influence the evolution or pathogenesis of IRDS and CLD. We can determine when particular cytokines are most abundant and determine the upstream regulators of these genes. Clearly, testing each gene within a cytokine network is an extremely arduous and potential time-wasting task. Thankfully, new technologies such as gene arrays have been developed. Gene arraying is the ultimate in multitasking because it allows the monitoring of gene expression of tens of thousands of genes in parallel, thereby gaining insight into complex disease-related pathways and interactions. This can be extremely useful in the discovery phase of a research project, which can lead to hypothesis generation and spawn novel approaches to complex heterogeneous diseases. Recently, several papers have reported using gene arrays to understand the gene environment in several models of lung injury. Specifically, the temporal gene expression patterns have been assessed in models of NiSO4-induced acute lung injury (41) and bleomycin-induced pulmonary fibrosis (42). Gene arrays have also been used to study the smoke and hydrogen peroxide effects on gene expression of bronchial epithelial cells (43) and, clinically, gene arrays have defined gene patterns, which differentiate normal lungs from those of patients with sporadic and familial primary pulmonary hypertensions (44). Evidently, gene array analysis may be a useful technology in understanding the pathobiology of distinct clinical phenotypes of IRDS and CLD.

Even though gene arrays are extremely powerful tools, they do not provide an in-depth or global picture of the spatial and temporal expression pattern of proteins and do not reveal the extent to which proteins are post-translationally modified. Ultimately, proteins mediate the responses in IRDS and CLD, and no change in gene expression does not necessarily mean that the product of that gene is not involved in the disease process. A prime example of this is NF-B. NF-B potentially has the ability to regulate numerous genes involved in the inflammatory response associated with IRDS and CLD. Normally NF-B is present in its inactive form in the cytoplasm by its association with a protein called inhibitory-kB (I-kB). Through various activation signals, I-kB becomes phosphorylated and degraded, which then allows NF-B to translocate to the nucleus, where it binds specific sequences in the promoter regions of inflammatory genes (32). Because the actions of NF-B are entirely post-translational, the use of microarrays will not help in elucidating where this inflammatory mechanism fits into the pathogenesis of IRDS or CLD. Therefore, a means to screen a wide variety of proteins needs to be employed to truly understand the complexities of IRDS and CLD. Proteome analysis is most commonly accomplished by the combination of two-dimensional gel electrophoresis and mass spectrometry. This approach can support expression profiling of several thousand proteins in multiple samples, but is time-consuming, labor-intensive, and requires significant technical expertise. This has prompted the development of several new strategies. One such strategy is the development of protein profiling arrays in which recognition molecules would be capable of binding individual proteins moieties with appropriate affinity and specificity and can be detected quantitatively. Within the lung field, proteomics have been used to study (i) the protein components involved in peribronchial fibrosis involved in asthma (45); (ii) differential protein expression in the nasal lavage of nonsmokers and smokers (46); and (iii) the effect of endothelin-1 stimulation on lung fibroblast cells (47). Perhaps the greatest benefit in utilizing proteomics in the study of IRDS and CLD is that one can use biologic samples that are not suitable for mRNA analysis, such as the BAL. Potentially, proteome analysis of BAL fluid of patients with IRDS and patients with CLD patients may reveal new lung disease markers, which may dictate new treatment strategies.

In the next decade, integrating genomic and proteomic approaches, as outlined in Figure 2, will unquestionably extend our ability to predict, diagnose, and treat infants at risk of developing IRDS and/or CLD. Hopefully this will dramatically reduce the high levels of morbidity and mortality and reduce the costs of treating these patients. Currently, survivors of IRDS with asthma and bronchopulmonary dysplasia consume 20 times more annualized dollars than unaffected children and 5.9% of all dollars spent on children from 0 to 18 yr of age (48).

View larger version (41K): [in this window] [in a new window]   Figure 2.   Process describing the utilization of advances in genomics and proteomics to understand cytokine networks in IRDS and CLD. Utilizing neonatal rats and mice, with or without genetic modification, the global picture of genes and protein changes associated with IRDS and CLD can be ascertained through transcriptomics and proteomics. These can be analyzed to select for genes and proteins, which could diagnose or treat individuals at risk of IRDS and CLD. Changes in target mRNAs and proteins can then be confirmed using standard laboratory techniques, and the function of these targets assessed in more traditional animal models of IRDS and CLD (such as the premature baboon or sheep) or analyzed in the more controlled environment of in vitro cell/organ cultures.

	Footnotes

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