Hyperoxia has deleterious effects on lung form and function; however,
the molecular events initiated by oxygen exposure remain unclear. We h
ypothesized that macrophages function as important intermediaries in t
he protective response of lung tissues after exposure to hyperoxia. Th
is hypothesis was tested by exposing cultured macrophages (RAW 264.7 c
ells) to hyperoxia for 24 h and then applying the conditioned medium f
rom these cells to cultured pulmonary epithelial cells or to pulmonary
microvascular endothelial cells. We observed that the expression of m
anganese superoxide dismutase mRNA increased in both target cell lines
, Therefore, we next hypothesized that exposure of these macrophages t
o hyperoxia results in a change in gene expression which could be dete
cted by differential display PCR (ddPCR), This hypothesis was tested b
y exposing RAW 264.7 cells to greater than or equal to 95% oxygen (or
normoxia) for 24 h, harvesting RNA, and performing ddPCR, A cDNA fragm
ent upregulated by hyperoxia was identified and reamplified. Verificat
ion of differential expression of mRNA was done by Northern analysis.
A mRNA which was reproducibly upregulated by hyperoxia, as well as by
lipopolysaccharide and interferon gamma, was identified. The different
ially expressed PCR product was cloned and sequenced, revealing a prod
uct with 99% identity to mouse urokinase mRNA We speculate that one fu
nction of pulmonary macrophages following a hyperoxic exposure is to s
ecrete urokinase. (C) 1998 Academic Press.