Neil B. Sweezey

A novel developmentally regulated gene in lung mesenchyme: homology to a tumor-derived trypsin inhibitor.

We used differential display-PCR (DD-PCR) to identify glucocorticoid-inducible genes that regulate lung development in late gestation. DD-PCR, a method to screen for differentially expressed genes, is based on a comparison of mRNAs isolated from a subset of two or more cell populations by analysis of RT-PCR products on DNA-sequencing gels. We isolated cDNA probes representing mRNAs expressed in primary cultures of rat lung fibroblasts, but not in epithelial cells, on fetal day 20. A day 20 glucocorticoid-treated fibroblast cDNA library was screened with a single probe to isolate the 3.1-kb cDNA late-gestation lung 1( LGL1; GenBank accession no. AF109674 ) encoding a deduced polypeptide of 188 amino acids. Northern analysis confirmed that LGL1is expressed in human, rat, and mouse fetal lungs, induced by glucocorticoid, developmentally regulated in fibroblasts but not detectable in epithelium. In situ hybridization confirmed LGL1 expression in the mesenchyme, but not in the epithelium, of fetal rat lung, kidney, and gut. The predicted LGL1 gene product (lgl1) showed 81% homology to P25TI, a polypeptide trypsin inhibitor recently identified in human glioblastoma and neuroblastoma cells but not detected in normal human tissues. Both lgl1 and P25TI belong to the CRISP family of cysteine-rich extracellular proteins. Trypsin is produced by both normal bronchial epithelial and lung adenocarcinoma cells. Although additional studies will be necessary to clearly establish a functional role for lgl1, we propose that lgl1 has a role in normal lung development that is likely to be via regulation of extracellular matrix degradation.

Glucocorticoid receptor mRNA and protein in fetal rat lung in vivo: modulation by glucocorticoid and androgen

Pulmonary glucocorticoid receptor (GR) is essential to timely preparation for the onset of breathing air at birth. We have previously used primary culture of late-gestation fetal rat lung cells to demonstrate differential regulation of GR by glucocorticoid depending on cell type. In this study, we hypothesized that the action of glucocorticoid on GR mRNA expression and protein elaboration in lung cells might be modulated by interactions present in vivo but not in primary culture. Given that male sex hormone (androgen) has an inhibitory effect on antenatal lung development, we also postulated that androgen would decrease antenatal lung GR. We report that antenatal maternal injection of the glucocorticoid dexamethasone (1 mg/kg) enhanced fetal lung cellular levels of GR mRNA and protein as assessed by in situ hybridization and immunocytochemistry (ICC), respectively. ICC was performed using polyclonal rabbit anti-human antibody that reacts with rat GR whether bound to ligand or not and does not interfere with GR binding to DNA. Levels of GR mRNA and protein were enhanced in cells throughout all areas of the lung tissue, suggesting that interactions occurring in intact tissue may override the previously reported direct inhibition by glucocorticoid of GR protein elaboration in isolated fetal rat lung epithelial cells. Furthermore, antenatal administration of the androgen 5alpha-dihydrotestosterone (0.2 mg/kg) reduced tissue levels of GR mRNA and protein, consistent with androgenic inhibition of antenatal lung development by decreasing GR. We conclude that glucocorticoids and androgens exert opposite effects on fetal lung GR.Pulmonary glucocorticoid receptor (GR) is essential to timely preparation for the onset of breathing air at birth. We have previously used primary culture of late-gestation fetal rat lung cells to demonstrate differential regulation of GR by glucocorticoid depending on cell type. In this study, we hypothesized that the action of glucocorticoid on GR mRNA expression and protein elaboration in lung cells might be modulated by interactions present in vivo but not in primary culture. Given that male sex hormone (androgen) has an inhibitory effect on antenatal lung development, we also postulated that androgen would decrease antenatal lung GR. We report that antenatal maternal injection of the glucocorticoid dexamethasone (1 mg/kg) enhanced fetal lung cellular levels of GR mRNA and protein as assessed by in situ hybridization and immunocytochemistry (ICC), respectively. ICC was performed using polyclonal rabbit anti-human antibody that reacts with rat GR whether bound to ligand or not and does not interfere with GR binding to DNA. Levels of GR mRNA and protein were enhanced in cells throughout all areas of the lung tissue, suggesting that interactions occurring in intact tissue may override the previously reported direct inhibition by glucocorticoid of GR protein elaboration in isolated fetal rat lung epithelial cells. Furthermore, antenatal administration of the androgen 5α-dihydrotestosterone (0.2 mg/kg) reduced tissue levels of GR mRNA and protein, consistent with androgenic inhibition of antenatal lung development by decreasing GR. We conclude that glucocorticoids and androgens exert opposite effects on fetal lung GR.

Reduced Viability of Mice with Lung Epithelial-Specific Knockout of Glucocorticoid Receptor

Glucocorticoid (GC)-responsive epithelial-mesenchymal interactions regulate lung development. The GC receptor (GR) mediates GC signaling. Mice lacking GR in all tissues die at birth of respiratory failure. To determine the specific need for epithelial GR in lung development, we bred triple transgenic mice that carry SPC/rtTA, tet-O-Cre, and floxed, but not wild-type, GR genes. When exposed to doxycycline in utero, triple transgenic (GRepi⁻) mice exhibit a Cre-mediated recombination event that inactivates the floxed GR gene in airway epithelial cells. Immunofluorescence confirmed the elimination of GR in Cre-positive airway epithelial cells of late gestation GRepi⁻ mice. Embryonic Day 18.5 pups had a relatively immature appearance with increased lung cellularity and increased pools of glycogen in the epithelium. Postnatal Day 0.5 pups had decreased viability. We used quantitative RT-PCR to demonstrate that specific elimination of epithelial immunoreactive GR in GRepi⁻ mice is associated with reduced mRNA expression for surfactant proteins (SPs) A, B, C, and D; β- and γ-ENaC; T1α; the 10-kD Clara cell protein (CCSP); and aquaporin 5 (AQP5). Western blots confirmed reduced levels of AQP5 protein. No reduction in the levels of the GR transport protein importin (IPO)-13 was observed. Our findings demonstrate a requirement for lung epithelial cell GR in normal lung development. We speculate that impaired epithelial differentiation, leading to decreased SPs, transepithelial Na, and liquid absorption at birth, may contribute to the reduced survival of newborn mice with suppressed lung epithelial GR.

Lgl1, a mesenchymal modulator of early lung branching morphogenesis, is a secreted glycoprotein imported by late gestation lung epithelial cells

Secreted glycoproteins serve a variety of functions related to cell-cell communication in developmental systems. We cloned LGL1, a novel glucocorticoid-inducible gene in foetal lung, and described its temporal and spatial localization in the rat. Disruption of foetal mesenchyme-specific LGL1 expression using antisense oligodeoxynucleotides, which was associated with a 50% decrease in lgl1 protein levels, inhibited airway epithelial branching in foetal rat gestational day 13 lung buds in explant culture. These findings suggested that lgl1 functions as a secreted signalling molecule. We now provide evidence supporting a role for lgl1 in mesenchymal-epithelial interactions that govern lung organogenesis. Lgl1 is a secreted glycoprotein with a conserved N-terminal secretory signal peptide. Using dual immunofluorescence, intracellular lgl1 was found to co-localize with markers of the Golgi apparatus and endoplasmic reticulum, consistent with its association with secretory vesicles. Using pulse-chase studies, we show that lgl1 is a stable protein with a half-life of 11.5 h. Furthermore, at gestational days 20 and 21 (term=22), foetal distal lung epithelial cells import lgl1 protein. Taken together, our findings support distinct roles for lgl1 as a mediator of glucocorticoid-induced mesenchymal-epithelial interactions in early and late foetal lung organogenesis.

The cystic fibrosis gender gap: Potential roles of estrogen

Cystic fibrosis (CF) is a complex, multi‐system, autosomal recessive disease predominantly affecting Caucasians that leads to vigorous airway inflammation and chronic respiratory infection, commonly with Pseudomonas aeruginosa. A variety of factors significantly modify the progression and severity of CF lung disease and the timing of the resulting mortality. We summarize here data indicating that there is in CF a female disadvantage in survival and morbidity, called the “CF gender gap”. Although controversy exists regarding the nature and relative importance of the various contributing mechanisms involved, gender affects the progression of CF disease with respect to lung infection, decline in pulmonary function and nutritional status. These interrelated factors in turn have a negative impact on survival. This review will emphasize the increasing evidence that suggest a role for the effects of gender, and particularly the female sex hormone estrogen, on infection, inflammation and transepithelial ion transport, all major determinants of CF lung disease. Future elucidation of the pathophysiology of hormonal aggravation of CF lung disease may pave the way for novel therapeutic interventions. This, combined with the magnitude of the gender gap in CF mortality, strongly suggests that further work in this field is well justified. Pediatr Pulmonol. 2014; 49:309–317.

Developmental differences of cystic fibrosis transmembrane conductance regulator functional expression in isolated rat fetal distal airway epithelial cells.

ABSTRACT: Fluid secretion from the pulmonary epithelium may play a significant role in determining intrauterine lung development. We used suspensions of distal pulmonary epithelial cells isolated from rat fetuses to assess a shift in secretory mechanisms occurring in the lung of this species during late gestation. The impact of cAMP on distal airway epithelial cells isolated from d 18 to d 21 rat fetuses was evaluated with measurements of cell volume and 36Cl efflux rates. At d 18, 8-Br-cAMP stimulated a volume reduction measured by electronic cell sizing that was prevented by the Cl− channel blocker anthracene-9-carboxylate (A-9C) and reflected in an increased rate of A-9C sensitive 36Cl efflux. Because the cystic fibrosis trans-membrane conductance regulator (CFTR) is thought to be a cAMP-regulated Cl− channel, we measured the effect of prior cell incubation with oligodeoxynucleotides antisense to the transcription site of the human CFTR gene on these events. We found that in antisense oligomer-treated cells, but not in sense oligomer-treated controls, volume and 36Cl efflux responses to 8-Br-cAMP were prevented in d 18 cells. In d 21 cells, 8-Br-cAMP did not stimulate volume reduction but the calcium ionophore A23187 did elicit cell volume reduction in cells suspended in an isotonic Ca2+-containing medium that was prevented by A-9C. This response to the ionophore was not found in the d 18 cells, and incubation with the antisense CFTR oligomer had no effect on the ionophore-induced responses in d 21 cells. Our results suggest that CFTR mediates cAMP-stimulated Cl− conductance in d 18 rat fetal distal pulmonary epithelial cells. Furthermore, during fetal growth between d 18 and 21 there is a reciprocal pattern whereby Ca2+-activated Cl− conductance appears whereas CFTR disappears in the distal pulmonary epithelium.

Intrinsic predisposition of naïve cystic fibrosis T cells to differentiate towards a Th17 phenotype

BackgroundCystic fibrosis (CF) is a complex, multi-system, life-shortening, autosomal recessive disease most common among Caucasians. Pulmonary pathology, the major cause of morbidity and mortality in CF, is characterized by dysregulation of cytokines and a vicious cycle of infection and inflammation. This cycle causes a progressive decline in lung function, eventually resulting in respiratory failure and death. The Th17 immune response plays an active role in the pathogenesis of CF pulmonary pathology, but it is not known whether the pathophysiology of CF disease contributes to a heightened Th17 response or whether CF naïve CD4+ T lymphocytes (Th0 cells) intrinsically have a heightened predisposition to Th17 differentiation.MethodsTo address this question, Th0 cells were isolated from the peripheral blood of CF mice, human CF subjects and corresponding controls. Murine Th0 cells were isolated from single spleen cell suspensions using fluorescence-activated cell sorting. Lymphocytes from human buffy coats were isolated by gradient centrifugation and Th0 cells were further isolated using a human naïve T cell isolation kit. Th0 cells were then assessed for their capacity to differentiate along Th17, Th1 or Treg lineages in response to corresponding cytokine stimulation. The T cell responses of human peripheral blood cells were also assessed ex vivo using flow cytometry.ResultsHere we identify in both mouse and human CF an intrinsically enhanced predisposition of Th0 cells to differentiate towards a Th17 phenotype, while having a normal propensity for differentiation into Th1 and Treg lineages. Furthermore, we identify an active Th17 response in the peripheral blood of human CF subjects.ConclusionsWe propose that these novel observations offer an explanation, at least in part, for the known increased Th17-associated inflammation of CF and the early signs of inflammation in CF lungs before any evidence of infection. Moreover, these findings point towards direct modulation of T cell responses as a novel potential therapeutic strategy for combating excessive inflammation in CF.

Functional Rescue of DeltaF508-CFTR by Peptides Designed to Mimic Sorting Motifs

The cystic fibrosis (CF)-causing mutant, deltaF508-CFTR, is misfolded and fails to traffic out of the endoplasmic reticulum (ER) to the cell surface. Introduction of second site mutations that disrupt a diarginine (RXR)-based ER retention motif in the first nucleotide binding domain rescues the trafficking defect of deltaF508-CFTR, supporting a role for these motifs in mediating ER retention of the major mutant. To determine if these RXR motifs mediate retention of the native deltaF508-CFTR protein in situ, we generated peptides that mimic these motifs and should antagonize mistrafficking mediated via their aberrant exposure. Here we show robust rescue of deltaF508-CFTR in cell lines and in respiratory epithelial tissues by transduction of RXR motif-mimetics, showing that abnormal accessibility of this motif is a key determinant of mistrafficking of the major CF-causing mutant.

Inflammatory cytokines, goblet cell hyperplasia and altered lung mechanics in Lgl1+/- mice

BackgroundNeonatal lung injury, a leading cause of morbidity in prematurely born infants, has been associated with arrested alveolar development and is often accompanied by goblet cell hyperplasia. Genes that regulate alveolarization and inflammation are likely to contribute to susceptibility to neonatal lung injury. We previously cloned Lgl1, a developmentally regulated secreted glycoprotein in the lung. In rat, O2 toxicity caused reduced levels of Lgl1, which normalized during recovery. We report here on the generation of an Lgl1 knockout mouse in order to determine whether deficiency of Lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury.MethodsAn Lgl1 knockout mouse was generated by introduction of a neomycin cassette in exon 2 of the Lgl1 gene. To evaluate the pulmonary phenotype of Lgl1+/- mice, we assessed lung morphology, Lgl1 RNA and protein, elastin fibers and lung function. We also analyzed tracheal goblet cells, and expression of mucin, interleukin (IL)-4 and IL-13 as markers of inflammation.ResultsAbsence of Lgl1 was lethal prior to lung formation. Postnatal Lgl1+/- lungs displayed delayed histological maturation, goblet cell hyperplasia, fragmented elastin fibers, and elevated expression of TH2 cytokines (IL-4 and IL-13). At one month of age, reduced expression of Lgl1 was associated with elevated tropoelastin expression and altered pulmonary mechanics.ConclusionOur findings confirm that Lgl1 is essential for viability and is required for developmental processes that precede lung formation. Lgl1+/- mice display a complex phenotype characterized by delayed histological maturation, features of inflammation in the post-natal period and altered lung mechanics at maturity. Lgl1 haploinsufficiency may contribute to lung disease in prematurity and to increased risk for late-onset respiratory disease.

Differential regulation of glucocorticoid receptor expression by ligand in fetal rat lung cells.

The glucocorticoid receptor (GR) mediates glucocorticoid stimulation of surfactant production by fetal mammalian lung. In many other tissues, glucocorticoids decrease expression of GR, thereby reducing responsiveness to these hormones. We therefore determined whether there is a similar effect of exogenous glucocorticoids on GR in fetal rat whole lung, and in the principal cell types involved in the stimulation of surfactant, the fibroblasts and the epithelial cells. The ontogeny of GR in late gestation lung differed between the two cell types, with maximal levels occurring in fibroblasts on gestational d 19, and on d 20 in epithelial cells. Administration of dexamethasone (1 mg/kg) to the mother on gestational d 18 or 19 (term = 22 d) increased specific GR binding activity in whole lung 24 h later. Furthermore, in vitro, incubation of cultured fibroblasts of gestational d 20 with 10−7 M Cortisol increased GR immunoreactive protein and binding activity in a dose- and time-dependent manner, without affecting cellular levels of GR mRNA. However, identical treatment of d 20 distal airway epithelial cells was followed by decreased GR protein without significant change in cellular GR mRNA. Surfactant protein-A protein levels, taken as assessments of lung maturation, were increased in response to the same treatment. Our findings suggest that hormonal regulation of GR in fetal lung cells occurs at a posttranscriptional level, and is cell-specific. In the context of substantial increases in circulating glucocorticoid concentrations during late gestation, these findings may be of physiologic importance to the biochemical maturation of the antenatal lung.