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 (PC) synthesis (2-fold) and higher saturation of PC (approximately 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.
Gonzales
L W
LW
Department of Pediatrics, Children's Hospital of Philadelphia, Pennsylvania, USA. gonzalesl@emial.chop.edu
Angampalli
S
S
Guttentag
S H
SH
Beers
M F
MF
Feinstein
S I
SI
Matlapudi
A
A
Ballard
P L
PL
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology*; D005938:Glucocorticoids / Q000378:metabolism / Q000494:pharmacology; D006023:Glycoproteins / Q000096:biosynthesis; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008168:Lung / Q000166:cytology / Q000196:embryology*; D008854:Microscopy, Electron; D010078:Oxazines / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D010969:Plastics; D011233:Precipitin Tests; D011510:Proteolipids / Q000096:biosynthesis; D037662:Pulmonary Surfactant-Associated Protein A; D037663:Pulmonary Surfactant-Associated Protein D; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D013501:Surface-Active Agents / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D004396:Coloring Agents; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D005938:Glucocorticoids; D006023:Glycoproteins; D010078:Oxazines; D010713:Phosphatidylcholines; D010726:Phosphodiesterase Inhibitors; D010743:Phospholipids; D010969:Plastics; D011510:Proteolipids; D037662:Pulmonary Surfactant-Associated Protein A; D037663:Pulmonary Surfactant-Associated Protein D; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D012333:RNA, Messenger; D013501:Surface-Active Agents; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003907:Dexamethasone; D000242:Cyclic AMP; D014357:Trypsin; D017364:Collagenases; C044808:nile red; D015056:1-Methyl-3-isobutylxanthine