Secretory activity and cell cycle alteration of alveolar type II cells in the early and late phase after irradiation.
OBJECTIVE
Type II cells and the surfactant system have been proposed to play a central role in pathogenesis of radiation pneumonitis. We analyzed the secretory function and proliferation parameters of alveolar type II cells in the early (until 24 h) and late phase (1-5 weeks) after irradiation (RT) in vitro and in vivo.


METHODS
Type II cells were isolated from rats according to the method of Dobbs. Stimulation of secretion was induced with terbutaline, adenosine triphosphate (ATP), and 12-O-tetradecanoylphorbol-13-acetate (TPA) for a 2-h period. Determination of secretion was performed using (3)H-labeled phosphatidylcholine. For the early-phase analysis, freshly isolated and adherent type II cells were irradiated in vitro with 9-21 Gy (stepwise increase of 3 Gy). Secretion stimulation was initiated 1, 6, 24, and 48 h after RT. For late-phase analysis, type II cells were isolated 1-5 weeks after 18 Gy whole lung or sham RT. Each experiment was repeated at least fivefold. Flow cytometry was used to determine cell cycle distribution and proliferating cell nuclear antigen index.


RESULTS
During the early-phase (in vitro) analysis, we found a normal stimulation of surfactant secretion in irradiated, as well as unirradiated, cells. No change in basal secretion and no dose effect were seen. During the late phase, 1-5 weeks after whole lung RT, we observed enhanced secretory activity for all secretagogues and a small increase in basal secretion in Weeks 3 and 4 (pneumonitis phase) compared with controls. The total number of isolated type II cells, as well as the rate of viable cells, decreased after the second post-RT week. Cell cycle alterations suggesting an irreversible G(2)/M block occurred in the second post-RT week and did not resolve during the observation period. The proliferating cell nuclear antigen index of type II cells from irradiated rats did not differ from that of controls.


CONCLUSIONS
In contrast to literature data, we observed no direct effect of radiation on secretory activity in the early phase after RT. In our study of isolated type II cells, as well as in intact animals, RT did not result in an impaired surfactant secretion up to 5 weeks after RT. Our in vivo experiments even showed an increased response of phosphatidylcholine secretion to all known secretagogues at Weeks 3 and 4 after whole lung RT, possibly due to inflammatory cytokines. Cell cycle alterations with G(2)/M block and cell loss in the late post-RT period may contribute more to the manifestation of radiation-induced lung damage than functional impairment in type II cells.
Willner
Jochen
J
Department of Radiotherapy, University of Wuerzburg, Wuerzburg, Germany. willner@strahlentherapie.uni-wuerzburg.de
Vordermark
Dirk
D
Schmidt
Michael
M
Gassel
Andreamaria
A
Flentje
Michael
M
Wirtz
Hubert
H
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002452:Cell Count; D002453:Cell Cycle / Q000502:physiology / Q000528:radiation effects*; D002455:Cell Division / Q000502:physiology / Q000528:radiation effects; D002470:Cell Survival / Q000502:physiology / Q000528:radiation effects; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008297:Male; D010713:Phosphatidylcholines / Q000032:analysis; D011650:Pulmonary Alveoli / Q000166:cytology* / Q000378:metabolism; D011663:Pulmonary Surfactants / Q000737:chemistry / Q000378:metabolism*; D017564:Radiation Pneumonitis / Q000209:etiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D015415:Biomarkers; D010713:Phosphatidylcholines; D011663:Pulmonary Surfactants; D007770:L-Lactate Dehydrogenase