Linda W. Gonzales

Functional and Trafficking Defects in ATP Binding Cassette A3 Mutants Associated with Respiratory Distress Syndrome

Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures.

IL-17A and TNF-α exert synergistic effects on expression of CXCL5 by alveolar type II cells in vivo and in vitro.

CXCL5, a member of the CXC family of chemokines, contributes to neutrophil recruitment during lung inflammation, but its regulation is poorly understood. Because the T cell-derived cytokine IL-17A enhances host defense by triggering production of chemokines, particularly in combination with TNF-α, we hypothesized that IL-17A would enhance TNF-α–induced expression of CXCL5. Intratracheal coadministration of IL-17A and TNF-α in mice induced production of CXCL1, CXCL2, and CXCL5, which was associated with increased neutrophil influx in the lung at 8 and 24 h. The synergistic effects of TNF-α and IL17A were greatly attenuated in Cxcl5−/− mice at 24 h, but not 8 h, after exposure, a time when CXCL5 expression was at its peak in wild-type mice. Bone marrow chimeras produced using Cxcl5−/− donors and recipients demonstrated that lung-resident cells were the source of CXCL5. Using differentiated alveolar epithelial type II (ATII) cells derived from human fetal lung, we found that IL-17A enhanced TNF-α–induced CXCL5 transcription and stabilized TNF-α–induced CXCL5 transcripts. Whereas expression of CXCL5 required activation of NF-κB, IL-17A did not increase TNF-α–induced NF-κB activation. Apical costimulation of IL-17A and TNF-α provoked apical secretion of CXCL5 by human ATII cells in a transwell system, whereas basolateral costimulation led to both apical and basolateral secretion of CXCL5. The observation that human ATII cells secrete CXCL5 in a polarized fashion may represent a mechanism to recruit neutrophils in host defense in a fashion that discriminates the site of initial injury.

Loss of Murine Na/myo-Inositol Cotransporter Leads to Brain myo-Inositol Depletion and Central Apnea*

myo-Inositol (Ins) and its polyphosphoinositide derivatives that are important in membrane signaling have long been held to play a special role in brain metabolism. As polyphosphoinositides turn over rapidly and are exceptionally abundant in nervous tissue, high Ins levels in the range of 2–15 mm that have been observed in brain may be necessary to maintain the rates of phosphoinositide synthesis in diverse membrane locations within neurons. Cellular concentration gradients of this magnitude indicate a dependence on active Ins transport, especially at the time of growth and differentiation. The Na+/myo-inositol cotransporter (SMIT1 or SLC5A3) gene is highly expressed prenatally in the central nervous system and placenta. To gain more insight into brain Ins metabolism, while ascertaining the importance of SMIT1 as a transporter, we generated mice with a homozygous targeted deletion of this gene. Newborn SMIT1(−/−) animals have no evidence of SMIT1 mRNA, a 92% reduction in the level of brain Ins, an 84% reduction in whole body Ins, and expire shortly after birth due to hypoventilation. Gross pathologic and light microscopic examinations of each organ, as well as the placenta, of embryonic day 18.5 fetuses at near term gestation were normal. Based on [3H]acetate incorporation into phospholipids of lung tissue explants, immunostaining of lung tissue for surfactant protein A, B, and C, and electron microscopic examination of alveolar cells, there was no evidence of abnormal pulmonary surfactant production by type 2 pneumocytes in lung. Although no histologic lesions were detected in the nervous system, electrophysiological studies of the brainstem pre-Bötzinger respiratory control center demonstrated an abnormal rhythm discharge with periods of central apnea. The cause of death can be explained by the regulatory defect in brainstem control of ventilation. This model demonstrates the critical importance ofSMIT1 in the developing nervous system. The high affinity SMIT1 transporter is responsible for the Ins concentration gradient in the murine fetal-placental unit.

Surfactant protein B in human fetal lung: developmental and glucocorticoid regulation.

ABSTRACT: Pulmonary surfactant protein B (SP-B) enhances phospholipid film formation in vitro and is essential for normal surfactant function in vivo. We examined human fetal lung before and during explant culture for content and cellular localization of SP-B mRNA and protein. SP-B mRNA was low in preculture specimens (18–20 wk) but hybridization signal increased over epithelial cells during culture and was enhanced by dexamethasone treatment (10 nM). SP-B immunofluorescence was very low in preculture specimens, increased during culture, and was uniformly intense in epithelial cells of dexamethasone-treated tissue. With a newly developed immunoassay, SP-B protein was undetectable in preculture lung (<2% of adult), appeared during culture (26% of adult), and was further increased ∼3-fold by dexamethasone treatment (86% of adult); lung tissue of two newborn infants contained 7–9-fold more SP-B than is found in the adult. Using Western blot with enhanced chemiluminescence, mature SP-B was undetectable in 16-wk specimens but was present in 19–24-wk preculture tissue at 0.2–2.9% of the adult level. By comparison, SP-B mRNA content is 14 and 50% of adult level in 19− and 24-wk lung tissue, respectively; levels increase 3-fold during culture and a further 3-fold with dexamethasone. Based on these observed differences between mRNA and protein content, we conclude that basal SP-B gene expression in epithelial cells of human fetal lung is regulated primarily at the level of translation or protein stability, whereas glucocorticoids act transcriptionally. We speculate that SP-B protein accumulates only as type II cells differentiate and acquire lamellar bodies for processing and storage of SP-B.

Surfactant composition and function in a primate model of infant chronic lung disease: Effects of inhaled nitric oxide

Bronchopulmonary dysplasia, or chronic lung disease (CLD), of premature infants involves injury from hyperoxia and mechanical ventilation to an immature lung. We examined surfactant and nitric oxide (NO), which are developmentally deficient in premature infants, in the baboon model of developing CLD. Fetuses were delivered at 125 d gestation and were managed for 14 d with ventilation and oxygen prn without (controls) or with inhaled NO at 5 ppm. Compared with term infants, premature control infants had reduced maximal lung volume, decreased tissue content of surfactant proteins SP-A, -B, and -C, abnormal lavage surfactant as assessed by pulsating bubble surfactometer, and a low concentration of SP-B/phospholipid. NO treatment significantly increased maximal lung volume and tissue SP-A and SP-C, reduced recovery of lavage surfactant by 33%, decreased the total protein:phospholipid ratio of surfactant by 50%, and had no effect on phospholipid composition or SP content except for SP-C (50%). In both treatment groups, levels of SP-B and SP-C in surfactant were negatively correlated with STmin, with a 5-fold greater SP efficiency for NO versus control animals. By contrast, lung volume and compliance were not correlated with surfactant function. We conclude that surfactant is often dysfunctional in developing CLD secondary to SP-B deficiency. NO treatment improves the apparent ability of hydrophobic SP to promote low surface tension, perhaps secondary to less protein inactivation of surfactant, and improves lung volume by a process unrelated to surfactant function.

TGF-β1 inhibits surfactant component expression and epithelial cell maturation in cultured human fetal lung

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine shown to play a critical role in organ morphogenesis, development, growth regulation, cellular differentiation, gene expression, and tissue remodeling after injury. We examined the effect of exogenously administered TGF-β1 on the expression of surfactant proteins (SPs) and lipids, fatty acid synthetase, and ultrastructural morphology in human fetal lung cultured for 5 days with and without dexamethasone (10 nM). Expression of the type II cell-specific marker surfactant proprotein C (proSP-C), studied by [35S]Met incorporation and immunoprecipitation, increased sevenfold with dexamethasone treatment. TGF-β1 (0.1-100 ng/ml) in the presence of dexamethasone inhibited 21-kDa proSP-C expression in a dose-dependent manner (maximal inhibition 31% of control level at 100 ng/ml). There was no change in [35S]Met incorporation into total protein in any of the treatment groups vs. the control group. In immunoblotting experiments, TGF-β1 blocked culture-induced accumulation of SP-A and SP-B. Under the same conditions, TGF-β1 reduced mRNA content for SP-A, SP-B, and SP-C to 20, 38, and 41%, respectively, of matched control groups but did not affect levels of β-actin mRNA. SP transcription rates after 24 h of exposure to TGF-β1 were reduced to a similar extent (20-50% of control level). In both control and dexamethasone-treated explants, TGF-β1 (10 ng/ml) also decreased fatty acid synthetase mRNA, protein, and enzyme activity and the rate of [3H]choline incorporation into phosphatidylcholine. By electron microscopy, well-differentiated type II cells lining potential air spaces were present in explants cultured with dexamethasone, whereas exposure to TGF-β1 with or without dexamethasone resulted in epithelial cells lacking lamellar bodies. We conclude that exogenous TGF-β1 disrupts culture-induced maturation of fetal lung epithelial cells and inhibits expression of surfactant components through effects on gene transcription.

Lamellar bodies of cultured human fetal lung: Content of surfactant protein A (SP-A), surface film formation and structural transformation in vitro

Lamellar bodies were isolated from dexamethasone and T3-treated explant cultures of human fetal lung, using sucrose density-gradient centrifugation. We examined their content of surfactant apoprotein A (SP-A), and their ability to form surface films and to undergo structural transformation in vitro. SP-A measured by ELISA composed less than 2% of total protein within lamellar bodies; this represented, as a minimum estimate, a 2-12-fold enrichment over homogenate. One- and two-dimensional gel electrophoresis also suggested that SP-A was a minor protein component of lamellar bodies. Adsorption of lamellar bodies to an air/water interface was moderately rapid, but accelerated dramatically upon addition of exogenous SP-A in ratios of 1:2-16 (SP-A:phospholipid, w/w). Similar adsorption patterns were seen for lamellar bodies from fresh adult rat and rabbit lung. Lamellar bodies incubated under conditions that promote formation of tubular myelin underwent structural rearrangement only in the presence of exogenous SP-A, with extensive formation of multilamellate whorls of lipid bilayers (but no classical tubular myelin lattices). We conclude that lamellar bodies are enriched in SP-A, but have insufficient content of SP-A for structural transformation to tubular myelin and rapid surface film formation in vitro.

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.

MAINTENANCE OF DIFFERENTIATED FUNCTION OF THE SURFACTANT SYSTEM IN HUMAN FETAL LUNG TYPE II EPITHELIAL CELLS CULTURED ON PLASTIC

We report a simplified culture system for human fetal lung type II cells that maintains surfactant expression. Type II cells isolated from explant cultures of hormone-treated lungs (18–22 wk gestation) by collagenase + trypsin digestion were cultured on plastic for 4 days in serum-free medium containing dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM) + isobutylmethylxanthine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs decreased markedly in control cells between days 1 and 4 of culture, but mRNA levels were high in treated cells on day 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increased SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greatest increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of phosphatidylcholine (PG) synthesis (2-fold) and higher saturation of PC (∼34% versus 27%) than controls. Only treated cells maintained secretagogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in isolated fetal type II cells, providing a simplified culture system for investigation of surfactant-related, and perhaps other, type II cell functions.

Glucocorticoid stimulation of fatty acid synthesis in explants of human fetal lung

We examined the effects of glucocorticoids and thyroid hormone (T3) on fatty acid synthesis, fatty acid composition and fatty acid synthetase activity in explants of human fetal lung (16-23 wk gestation). Explants were cultured 1-7 days in the absence (control) or presence of dexamethasone (10 nM) and/or T3 (2 nM). In control explants fatty acid synthesis and fatty acid synthetase activity increased 200% and 455%, respectively, between 1 and 5 days. Dexamethasone (10 nM) stimulated fatty acid synthesis (tritiated water incorporation) 155% and fatty acid synthetase activity 117% after 5 days in culture. T3 (2 nM) was not stimulatory, either alone or in the presence of dexamethasone. Dexamethasone increased the proportion of newly synthesized fatty acid recovered in phosphatidylcholine from 72% (control) to 90% (P less than 0.02) of total fatty acid. Dexamethasone stimulation of fatty acid synthetase activity was consistent with a receptor-mediated process: (1) stimulation was saturable and dose-dependent (Kd = 1.5 +/- 0.3 nM); (2) the potency of glucocorticoid analogs and other steroids reflected their glucocorticoid activity; (3) stimulation was reversible when cortisol was removed from the medium. Stimulation by dexamethasone was apparent within 24 h of hormone exposure, and increased to a maximum between 4 and 6 days. Fatty acid synthetase activity was higher in Type II cells (3.54 +/- 0.58 nmol malate/min per mg protein) than in fibroblasts from treated explants. Although both cell types responded to hormone treatment the stimulation was greater for Type II cells (200% vs. 75% increase). The fatty acid composition of PC showed increases in 14:0 and 16:1 with culture alone which were further stimulated by dexamethasone but not T3. These results indicate glucocorticoid stimulation of fatty acid synthesis and are consistent with a key role for fatty acid synthetase in the hormonal induction of pulmonary surfactant phosphatidylcholine synthesis in cultured fetal lung.