Linda Gonzales

Isolation and characterization of differentiated alveolar type II cells from fetal human lung

A method has been developed for isolating differentiated type II cells from human lung of 18-24-week gestation. The procedure involves an initial 4-day culture of lung explants in the presence of dexamethasone (10 nM) and triiodothyronine (2 nM). Type II cells (and fibroblasts) are isolated by trypsin digestion of the explants, two differential adherence steps and incubation overnight in primary culture. This method provides a high yield of type II cells ((50 +/- 15) X 10(6) cells/g wet weight of explant) with a purity of 85 +/- 5% in 16 experiments. The type II cells contain numerous perinuclear granules which stain darkly with toluidine blue and Papanicolaou stain; electron microscopy showed these inclusions to be lamellar bodies with tightly stacked, well defined lamellae. Type II cells, but not fibroblasts, were positive by immunofluorescence histology for surfactant apoprotein and binding of Maclura pomifera lectin which binds to the surface of type II but not type I cells in vivo. The rate of both [3H]acetate and [3H]choline incorporation into phosphatidylcholine (PC) was several-fold greater in type II cells than fibroblasts; the saturation of PC was 36.2 and 25.9%, respectively. Release of saturated PC was stimulated by terbutaline, the ionophore A23187, and tetradecanoyl phorbol acetate in type II cells but not fibroblasts. We conclude that differentiated type II cells can be isolated in relatively high yield and purity from hormone-treated explants of fetal human lung.

Early Alveolar Epithelial Dysfunction Promotes Lung Inflammation in a Mouse Model of Hermansky-Pudlak Syndrome

RATIONALE The pulmonary phenotype of Hermansky-Pudlak syndrome (HPS) in adults includes foamy alveolar type 2 cells, inflammation, and lung remodeling, but there is no information about ontogeny or early disease mediators. OBJECTIVES To establish the ontogeny of HPS lung disease in an animal model, examine disease mediators, and relate them to patients with HPS1. METHODS Mice with mutations in both HPS1/pale ear and HPS2/AP3B1/pearl (EPPE mice) were studied longitudinally. Total lung homogenate, lung tissue sections, and bronchoalveolar lavage (BAL) were examined for phospholipid, collagen, histology, cell counts, chemokines, surfactant protein D (SP-D), and S-nitrosylated SP-D. Isolated alveolar epithelial cells were examined for expression of inflammatory mediators, and chemotaxis assays were used to assess their importance. Pulmonary function test results and BAL from patients with HPS1 and normal volunteers were examined for clinical correlation. MEASUREMENTS AND MAIN RESULTS EPPE mice develop increased total lung phospholipid, followed by a macrophage-predominant pulmonary inflammation, and lung remodeling including fibrosis. BAL fluid from EPPE animals exhibited early accumulation of both SP-D and S-nitrosylated SP-D. BAL fluid from patients with HPS1 exhibited similar changes in SP-D that correlated inversely with pulmonary function. Alveolar epithelial cells demonstrated expression of both monocyte chemotactic protein (MCP)-1 and inducible nitric oxide synthase in juvenile EPPE mice. Last, BAL from EPPE mice and patients with HPS1 enhanced migration of RAW267.4 cells, which was attenuated by immunodepletion of SP-D and MCP-1. CONCLUSIONS Inflammation is initiated from the abnormal alveolar epithelial cells in HPS, and S-nitrosylated SP-D plays a significant role in amplifying pulmonary inflammation.

Corticosteroid binding by fetal rat and rabbit lung in organ culture

To further characterize glucocorticoid action in fetal lung cells, we investigated corticosteroid metabolism and binding in explants of fetal rat and rabbit lung. Cortisone (E) was concerted to cortisol (F) and bound by receptor with a time course only somewhat slower than for F. Production of F (0.243 pmol/min/mg DNA) was the same in male and female rabbits and was not affected by prior exposure to glucocorticoid in utero or in culture. The t 1/2 for dissociation of nuclear-bound [3H]F was 84 min on changing the culture medium and 21 min on addition of excess non-labeled dexamethasone. Dissociation of [3H]dexamethasone was approx 5-fold slower by both procedures. The KD for nuclear binding of dexamethasone, F, E, and corticosterone in rabbit lung were 0.7, 7.3, 6.8 and 70.6 nM, respectively. In rat lung, the KD for dexamethasone was 6.8 nM. The concentrations of dexamethasone and F required for half-maximal stimulation of phosphatidylcholine synthesis were similar to the KD values. Dexamethasone binding capacity (sites/mg DNA) increased with age in both rat (+103% increase from day 16 to 22) and rabbit (+47% between day 23 and 30). Receptor concentration was the same in both sexes, and there were no developmental changes in non-specific binding, nuclear:cytoplasmic distribution, or KD. In 27-day rabbit fetuses, the rate of choline incorporation was higher in lungs with greater binding capacity. We conclude that (1) E is rapidly converted to F in rabbit lung to become an active glucocorticoid, whereas corticosterone probably has little physiologic activity, (2) there is a species difference in the affinity of dexamethasone binding which is reflected in responsiveness (3) there is no difference between sexes in E conversion, receptor capacity, or phosphatidylcholine synthesis, and (4) the concentration of binding sites per lung cell increases during fetal development. We suggest that developmental increases in both F production and receptor may be important factors in the expression of endogenous glucocorticoid effects.

Analysis of a lung defect in autophagy-deficient mouse strains

Yeast Atg1 initiates autophagy in response to nutrient limitation. The Ulk gene family encompasses the mammalian orthologs of yeast ATG1. We created mice deficient for both Ulk1 and Ulk2 and found that the mice die within 24 h of birth. When found alive, pups exhibited signs of respiratory distress. Histological sections of lungs of the Ulk1/2 DKO pups showed reduced airspaces with thickened septae. A similar defect was seen in Atg5-deficient pups as both Ulk1/2 DKO and Atg5 KO lungs show numerous glycogen-laden alveolar type II cells by electron microscopy, PAS staining, and increased levels of glycogen in lung homogenates. No abnormalities were noted in expression of genes encoding surfactant proteins but the ability to incorporate exogenous choline into phosphatidylcholine, the major phospholipid component of surfactant, was increased in comparison to controls. Despite this, there was a trend for total phospholipid levels in lung tissue to be lower in Ulk1/2 DKO and Atg5 KO compared with controls. Autophagy was abundant in lung epithelial cells from wild-type mice, but lacking in Atg5 KO and Ulk1/2 DKO mice at P1. Analysis of the autophagy signaling pathway showed the existence of a negative feedback loop between the ULK1 and 2 and MTORC1 and 2, in lung tissue. In the absence of autophagy, alveolar epithelial cells are unable to mobilize internal glycogen stores independently of surfactant maturation. Together, the data suggested that autophagy plays a vital role in lung structural maturation in support of perinatal adaptation to air breathing.

HTII-280, a Biomarker Specific to the Apical Plasma Membrane of Human Lung Alveolar Type II Cells

The pulmonary alveolar epithelium is composed of two morphologically distinct cell types, type I (TI) and type II (TII) cells. Alveolar TII cells synthesize, secrete, and recycle surfactant components; contain ion transporters; and secrete immune effector molecules. In response to alveolar injury. TII cells have the capacity to act as progenitor cells, proliferating and transdifferentiating into TI cells. Although various proteins are associated with TII cells, a plasma membrane marker specific to human TII cells that would be useful for identification in tissue and for isolating this cell type has not been described previously. We devised a strategy to produce a monoclonal antibody (MAb) specific to the apical surface of human TII cells and developed an MAb that appears to be specific for human TII cells. The antibody recognizes a 280- to 300-kDa protein, HTII-280, which has the biochemical characteristics of an integral membrane protein. HTII-280 is detected by week 11 of gestation and is developmentally regulated. HTII-280 is useful for isolating human TII cells with purities and viabilities >95%. HTII-280 is likely to be a useful morphological and biochemical marker of human TII cells that may help to advance our understanding of various lung pathological conditions, including the origin and development of various lung tumors.

|[beta]|-Adrenergic Receptors and cAMP Response Increase during Explant Culture of Human Fetal Lung: Partial Inhibition by Dexamethasone

ABSTRACT: We studied β-adrenergic receptors and responses in human fetal lung (15–25 wk gestation) maintained in explant culture with and without added dexamethasone. To determine β-adrenergic receptor concentration, we performed radioligand binding assays with [125I]-iodocyanopindolol. We also examined the ability of isoproterenol to stimulate cAMP generation as a measure of response to β-adrenergic receptor occupancy. In control cultures, β-receptor concentration increased significantly from d 0 to 3 of culture and thereafter remained stable. The kd (∼24 pM) of [125I]-iodocyanopindolol did not change with time in culture. The ability of isoproterenol to stimulate cAMP generation over basal levels increased in controls throughout the 5 d in explant culture. Addition of dexamethasone (10 nM) to the culture medium partially blocked the increase in β-receptor concentration and decreased both cAMP content and generation (basal and stimulated) in a dose-dependent manner (median effective concentration ±1 nM). In these same explants, dexamethasone increased the activity of fatty acid synthetase, an enzyme important in surfactant synthesis, more than 2-fold. Our results indicate that β-adrenergic receptors and isoproterenol stimulation of cAMP generation increase spontaneously in human fetal lung grown in explant culture. Dexamethasone, which accelerates other aspects of human lung development in vitro, decreases β-adrenergic receptor concentration and inhibits β-adrenergic responses.

GLUCOCORTICOID AND THYROID HORMONE STIMULATION OF PHOSPHATIDYLCHOLINE |[lpar]|PC|[rpar]| SYNTHESIS IN CULTURED HUMAN FETAL LUNG

We examined effects of dexamethasone (DEX) and T3 on PC synthesis in 40 specimens of human fetal lung (15-22 wk gestation) cultured for 4-8 d in serum-free Waymouths medium with 95% air/5% CO2 on a rocker platform. In the absence of hormones, there was no consistent change in choline incorporation into PC and saturated PC during culture; PC content was 18.3 ± 2.3 μg PC-Pi/mg DNA in preculture tissue and 39.4 ± 3.4 (n=13) after 7 d culture. Choline into PC was stimulated by T3, DEX, and T3+DEX 11.9 ± 4.8, 36.6 ± 11.7, and 107 ± 36.5% (n=7), respectively, after 2 d exposure, and 51.6 ± 12.6, 103.4 ± 14.2, and 165 ± 16.8% after 6 d exposure. The content of PC increased 37.0 ± 7.6, 90.3 ± 16, and 125 ± 22% after 6 d with T3, DEX, and T3+DEX, respectively. The % saturation of newly synthesized PC was 19.9 ± 1.8, 20.9 ± 2.1, 26.4 ± 1.5 (P < .05), and 26.0 ± 1.8 (P < .05) in control, T3-, DEX-, and T3+DEX-treated cultures, respectively. Stimulation occurred by 24 h and was optimal after 4-6 d of exposure to the hormones. DEX was optimal at ˜10 nM and T3 at ˜1 nM. Cortisol was about 10% as potent as DEX and estriol was inactive. T4 and rT3 were about 10% and 1% as potent as T3.We conclude that both DEX and T3 increase saturated PC synthesis in cultured human lung, apparently acting through receptor-mediated mechanisms. We suggest that combined hormonal treatment may be more effective than glucocorticoid alone in stimulating surfactant synthesis.

233 INTERACTION OF GLUCOCORTICOID, THYROID HORMONE AND PROLACTIN IN STIMULATION OF PHOSPHATIDYLCHOLINE (PC) SYNTHESIS IN CULTURED FETAL LUNG

To investigate hormonal interactions in regulation of surfactant synthesis, we assayed PC synthesis in organ cultures of fetal rat (18 d), rabbit (23 d) and human (17-25 wk) lung maintained in serum-free Waymouths medium for 2-6 d. Explants were exposed to no hormones, dexamethasone (Dex, 10-100 nM), T3 (2 nM), prolactin (PRL, 0.2-2 μg/ml) or combinations thereof. PC synthesis was assayed by incorporation of 3H-choline into PC for 4 h in 3-11 experiments. Results (mean±SE) are shown below.In contrast to published reports for rat and human lung, we found no significant effect by PRL either alone or in the presence of other hormones. Results were equivalent for 2 sources of PRL (NIH & Sigma), a range of exposure times (1-6 d), a range of doses (0.001-8 μg/ml), incorporation rate of 3H-acetate into PC and distribution of cpm among phospholipids (rabbit), incorporation of choline into saturated PC (rat), and rocker vs lens paper/grid culture systems (human). We conclude that Dex and T3 have optimal effects on surfactant synthesis in cultured fetal lung in the absence of exogenous PRL.

1395 HORMONES AND SURFACTANT SYNTHESIS IN EXPLANTS OF HUMAN FETAL LUNG

To further study the effects of glucocorticoids and thyroid hormones in fetal lung, we cultured human lung (16-22wk) for 4-8d as explants in serum-free Waymouths medium with 95% air/5% CO2. Effects of dexamethasone (Dex, 10 nM) and T3 (2 nM) on phosphatidylcholine (PC) synthesis varied with precursor (Table).The additive hormonal effect occurred over a range of choline concentrations, did not alter the distribution of label among acid-soluble precursors, and correlated with tissue saturated PC content. Dex, but not T3, altered the distribution of precursor among phospholipids; compared to controls, glycerol incorporated more into PG (13.5 vs 4.9%) and less into PI (13.1 vs 18.9%), and acetate incorporated more into PC (81.5 vs 73.1%) and less into sphingomyelin (2.1 vs 6.3%). By electron microscopy, epithelial cells of treated explants showed less glycogen, many mo amellar bodies, and proliferation of microvilli. We conclude that low concentrations of glucocorticoids and thyroid hormone stimulate surfactant production in fetal lung in the absence of serum or other hormones. The two hormones appear to act at different biochemical sites to produce a synergistic response.

291 ROLE OF |[beta]|-ADRENERGIC RECEPTORS (BAR) AND ENDOGENENOUS CATECHOLAMINES IN SURFACTANT RELEASE BUT NOT LUNG WATER ABSORPTION IN FETAL RABBITS

Exogenous β-agonists stimulate release of surfactant (SAM) and absorption of lung water, presumably through interaction with BAR. To evaluate the role of endogenous catecholamines in these processes, we injected fetuses (28-d gestation) with an irreversible BAR antagonist, bromacyl- (1-8) diamino-β-menthone alprenolol (B, 30-40 mg/kg) or vehicle. BAR concentration was reduced from 215±11 to 46±16 fmol/mg protein, measured by [125I] Cyanopindolol binding to lung particulate, with no change in KD or adenylate cyclase activity not mediated by BAR. SAM in lavage was decreased ∼30% in 2 nonbreathing groups (Table).B appears to inhibit SAM release per se rather than synthesis since treatment did not affect tissue saturated phosphatidyl-choline, [3H] choline incorporation by lung minces, or phospholipid composition of lavage. In studies of lung water, B did not attact the decreases observed after vaginal vs c/s (no labor) delivery or after birth. We conclude that endogenous catecholamines participate in SAM but not water flux in the fetus.