Pulmonary IL-1ß expression in early life causes permanent changes in lung structure and function in adulthood.

Chorioamnionitis, mechanical ventilation, oxygen therapy, and postnatal infection promote inflammation in the newborn lung. The long-term consequences of pulmonary inflammation during infancy have not been well characterized. The aim of this study was to examine the impact of inflammation during the late saccular to alveolar stages of lung development on lung structure and function in adulthood. To induce IL-1ß expression in the pulmonary epithelium of mice with a tetracycline-inducible human IL-1ß transgene, doxycycline was administered via intraperitoneal injections to bitransgenic pups and their littermate controls on postnatal days (PN) 0, 0.5, and 1. Lung structure, inflammation, and airway reactivity were studied in adulthood. IL-1ß production in early life resulted in increased numbers of macrophages and neutrophils on PN21, but inflammation subsided by PN42. Permanent changes in alveolar structure, i.e., larger alveoli and thicker alveolar walls, were present from PN21 to PN84. Lack of alveolar septation thus persisted after IL-1ß production and inflammation had ceased. Early IL-1ß production caused goblet cell hyperplasia, enhanced calcium-activated chloride channel 3 (CLCA3) protein expression, and increased airway reactivity in response to methacholine on PN42. Lymphoid follicles were present adjacent to small airways in the lungs of adult bitransgenic mice, and levels of the B cell chemoattractant CXC-motif ligand (CXCL) 13 were elevated in the lungs of bitransgenic mice compared with controls. In conclusion, IL-1ß-induced pulmonary inflammation in early life causes a chronic lung disease in adulthood.

IL-1ß expression in the distal lung epithelium disrupts lung morphogenesis and epithelial cell differentiation in fetal mice.

Perinatal inflammation and the inflammatory cytokine IL-1 can modify lung morphogenesis. To examine the effects of antenatal expression of IL-1ß in the distal airway epithelium on fetal lung morphogenesis, we studied lung development and surfactant expression in fetal mice expressing human IL-1ß under the control of the surfactant protein (SP)-C promoter. IL-1ß-expressing pups suffered respiratory failure and died shortly after birth. IL-1ß caused fetal lung inflammation and enhanced the expression of keratinocyte-derived chemokine (KC/CXCL1) and monocyte chemoattractant protein 3 (MCP-3/CCL7), the calgranulins S100A8 and S100A9, the acute-phase protein serum amyloid A3, the chitinase-like proteins Ym1 and Ym2, and pendrin. IL-1ß decreased the percentage of the total distal lung area made up of air saccules and the number of air saccules in the lungs of fetal mice. IL-1ß inhibited the expression of VEGF-A and its receptors VEGFR-1 and VEGFR-2. The percentage of the cellular area of the distal lung made up of capillaries was decreased in IL-1ß-expressing fetal mice. IL-1ß suppressed the production of SP-B and pro-SP-C and decreased the amount of phosphatidylcholine and the percentage of palmitic acid in the phosphatidylcholine fraction of lung phospholipids, indicating that IL-1ß prevented the differentiation of type II epithelial cells. The production of Clara cell secretory protein in the nonciliated bronchiolar (Clara) cells was likewise suppressed by IL-1ß. In conclusion, expression of IL-1ß in the epithelium of the distal airways disrupted the development of the airspaces and capillaries in the fetal lung and caused fatal respiratory failure at birth.

Transgenic overexpression of interleukin-1ß induces persistent lymphangiogenesis but not angiogenesis in mouse airways.

These studies used bi-transgenic Clara cell secretory protein (CCSP)/IL-1ß mice that conditionally overexpress IL-1ß in Clara cells to determine whether IL-1ß can promote angiogenesis and lymphangiogenesis in airways. Doxycycline treatment induced rapid, abundant, and reversible IL-1ß production, influx of neutrophils and macrophages, and conspicuous and persistent lymphangiogenesis, but surprisingly no angiogenesis. Gene profiling showed many up-regulated genes, including chemokines (Cxcl1, Ccl7), cytokines (tumor necrosis factor α, IL-1ß, and lymphotoxin-ß), and leukocyte genes (S100A9, Aif1/Iba1). Newly formed lymphatics persisted after IL-1ß overexpression was stopped. Further studies examined how IL1R1 receptor activation by IL-1ß induced lymphangiogenesis. Inactivation of vascular endothelial growth factor (VEGF)-C and VEGF-D by adeno-associated viral vector-mediated soluble VEGFR-3 (VEGF-C/D Trap) completely blocked lymphangiogenesis, showing its dependence on VEGFR-3 ligands. Consistent with this mechanism, VEGF-C immunoreactivity was present in some Aif1/Iba1-immunoreactive macrophages. Because neutrophils contribute to IL-1ß-induced lung remodeling in newborn mice, we examined their potential role in lymphangiogenesis. Triple-transgenic CCSP/IL-1ß/CXCR2(-/-) mice had the usual IL-1ß-mediated lymphangiogenesis but no neutrophil recruitment, suggesting that neutrophils are not essential. IL1R1 immunoreactivity was found on some epithelial basal cells and neuroendocrine cells, suggesting that these cells are targets of IL-1ß, but was not detected on lymphatics, blood vessels, or leukocytes. We conclude that lymphangiogenesis triggered by IL-1ß overexpression in mouse airways is driven by VEGF-C/D from macrophages, but not neutrophils, recruited by chemokines from epithelial cells that express IL1R1.

Role of CXC chemokine receptor-2 in a murine model of bronchopulmonary dysplasia.

The contribution of neutrophils and CXC chemokines to the pathogenesis of bronchopulmonary dysplasia is not well defined. The transgenic expression of IL-1ß in the pulmonary epithelium causes lung inflammation and disrupts alveolar development in infant mice. To study the hypothesis that CXC chemokine receptor-2 (CXCR2) is a mediator of inflammatory lung injury, we compared lung development in IL-1ß-expressing mice with wild-type (IL-1ß/CXCR2(+/+)) or null (IL-1ß/CXCR2(-/-)) CXCR2 loci. CXCR2 deficiency abolished the transmigration of neutrophils into the alveolar lumen in IL-1ß-expressing mice, but did not alter the number of neutrophils in the parenchyma. The deletion of CXCR2 increased the alveolar chord length and reduced the survival of mice when IL-1ß was expressed from the pseudoglandular to the alveolar stages. The capillary configuration was highly abnormal in both IL-1ß/CXCR2(+/+) and IL-1ß/CXCR2(-/-) lungs, but in very different ways. The cellular area of the parenchyma and the total capillary area of IL-1ß/CXCR2(+/+) and IL-1ß/CXCR2(-/-) mice were smaller than those of control/CXCR2(+/+) and control/CXCR2(-/-) mice, but the ratio of capillary area to cellular area was similar in all four genotypes. When IL-1ß was expressed during the saccular stage, IL-1ß/CXCR2(-/-) mice had smaller alveolar chord lengths and better survival than did IL-1ß/CXCR2(+/+) mice. Independent of the timing of IL-1ß expression, IL-1ß increased alveolar septal thickness in mice with wild-type CXCR2 loci, but not in CXCR2 null mice. Depending on the developmental stage at the time of the inflammatory insult, inhibition of the CXCR2 pathway may exert opposite effects on alveolar septation in the neonatal lung.

Developmental stage is a major determinant of lung injury in a murine model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a common inflammatory lung disease in premature infants. To study the hypothesis that the sensitivity of the lung to inflammatory injury depends on the developmental stage, we studied postnatal lung development in transgenic mice expressing human IL-1ß (hIL-1ß) in the lungs during the late canalicular-early saccular, saccular, or late saccular-alveolar stage. Overexpression of hIL-1ß in the saccular stage caused arrest in alveolar development, airway remodeling, and goblet cell hyperplasia in the lungs as well as poor growth and survival of infant mice. Overexpression of hIL-1ß during the late canalicular-early saccular stage did not adversely affect lung development, growth, or survival of the pups. Mice expressing hIL-1ß from the late saccular to alveolar stage had smaller alveolar chord length, thinner septal walls, less airway remodeling and mucus metaplasia, and better survival than mice expressing hIL-1ß during the saccular stage. Human IL-1ß overexpression in the saccular stage was sufficient to cause a BPD-like illness in infant mice, whereas the lung was more resistant to hIL-1ß-induced injury at earlier and later developmental stages.

Mechanisms of inflammatory lung injury in the neonate: lessons from a transgenic mouse model of bronchopulmonary dysplasia.

The role of inflammation in the pathogenesis of bronchopulmonary dysplasia (BPD) is not well understood. By using a transgenic mouse expressing the inflammatory cytokine interleukin (IL)-1beta in the lung, we have shown that perinatal expression of IL-1beta causes a BPD-like illness in infant mice. We have used this model to identify mechanisms by which inflammation causes neonatal lung injury. Increased matrix metalloproteinase (MMP)-9 activity is associated with BPD. MMP-9 deficiency worsens alveolar hypoplasia in IL-1beta-expressing newborn mice, suggesting that MMP-9 has a protective role in neonatal inflammatory lung injury. The beta6 integrin subunit, an activator of transforming growth factor-beta, is involved in adult lung disease. Absence of the beta6 integrin subunit improves alveolar development in IL-1beta-expressing mice, suggesting that the beta6 integrin subunit is a pathogenetic factor in inflammatory lung disease in the newborn. The authors of clinical studies who have examined maternal inflammation as a risk factor for BPD have found variable results. We have shown that maternal IL-1beta production preceding fetal IL-1beta production prevents lung inflammation, alveolar hypoplasia, and airway remodeling in newborn IL-1beta-expressing mice. Thus, maternal inflammation may protect the newborn lung against subsequent inflammatory injury. In contrast, when maternal and fetal production of IL-1beta are induced simultaneously, the development of IL-1beta-induced lung disease in the newborn is not prevented.

beta6 Integrin subunit deficiency alleviates lung injury in a mouse model of bronchopulmonary dysplasia.

Pulmonary inflammation is associated with the development of bronchopulmonary dysplasia in premature infants. We have previously shown that perinatal pulmonary expression of human IL-1beta is sufficient to cause a lung disease similar to bronchopulmonary dysplasia, characterized by inflammation, impaired alveolarization, poor postnatal growth, and increased mortality in infant mice. The alphavbeta6 integrin plays a critical role in regulating inflammation in the adult lung. To study the role of the beta6 integrin subunit in neonatal inflammatory lung disease, we compared the pulmonary development in IL-1beta-expressing infant mice with wild-type or null beta6 integrin loci. Absence of the beta6 integrin subunit decreased the mortality and improved the postnatal growth of IL-1beta-expressing pups. The disrupted alveolar development of IL-1beta-expressing mice was improved by beta6 integrin deficiency. IL-1beta-expressing beta6(-/-) pups had shorter alveolar chord length and thinner alveolar walls than IL-1beta-expressing beta6(+/+) pups. In addition, the absence of the beta6 integrin subunit reduced IL-1beta-induced neutrophil and macrophage infiltration into the alveolar spaces. beta6 integrin subunit deficiency suppressed inflammation and goblet cell hyperplasia in the airways and alleviated airway remodeling in IL-1beta-expressing mice. The expression of the chemoattractant proteins, keratinocyte-derived chemokine, macrophage-inflammatory protein-2, calgranulin A, and calgranulin B, of osteopontin, and of the chitinase-like lectins, Ym1 and Ym2, was lower in IL-1beta-expressing beta6(-/-) than in IL-1beta-expressing beta6(+/+) mice. We conclude that absence of the beta6 integrin subunit protects the infant murine lung against IL-1beta-induced inflammation and injury.

Matrix metalloproteinase-9 deficiency worsens lung injury in a model of bronchopulmonary dysplasia.

Increased activity of matrix metalloproteinase (MMP)-9 is associated with the development of bronchopulmonary dysplasia (BPD) in newborn infants, but the role of MMP-9 in the pathophysiology of BPD is unclear. We have shown that perinatal expression of interleukin-1 beta (IL-1 beta) in the lung is sufficient to cause a BPD-like illness in infant mice. To study the hypothesis that MMP-9 is an important downstream mediator in IL-1 beta-induced lung injury in the newborn, we compared the effects of IL-1 beta on fetal and postnatal lung inflammation and development in transgenic mice with regulatable pulmonary overexpression of human mature IL-1 beta with wild-type (IL-1 beta/MMP-9(+/+)) or null (IL-1 beta/MMP-9(-/-)) MMP-9 loci. IL-1 beta increased the expression of MMP-9 mRNA and amount of MMP-9 protein in the lungs of MMP-9(+/+) mice. IL-1 beta/MMP-9(-/-) mice had fewer neutrophils but more macrophages in the lungs than did IL-1 beta/MMP-9(+/+) mice. MMP-9 deficiency increased pulmonary cell death and macrophage clearance of dying cells in IL-1 beta-expressing mice. IL-1 beta/MMP-9(-/-) mice had more severe alveolar hypoplasia than IL-1 beta/MMP-9(+/+) mice, implying that IL-1 beta-induced lung disease was worsened in the absence of MMP-9. These results suggest that MMP-9 activity in the inflamed neonatal lung protects the lung against injury.