Late upregulation of VCAM-1 and ICAM-1 in irradiated murine pulmonary endothelial and lung parenchymal cells precedes recruitment of bone marrow-derived macrophages and fibrosis

Purpose: C57BL/6J mice develop organizing alveolitis/fibrosis at around 140 days after 20 Gy irradiation to both lungs. The cellular and molecular mechanism of late irradiation changes is unknown. To determine whether proliferation of a slowly cycling cell population was responsible for late increase in TGF-β1 and TGF-β2 detected at 100 to 120 days after irradiation, and whether upregulation of adhesion molecules was associated with macrophage and fibroblast proliferation in fibrotic lesions, we examined the pulmonary biochemical and cellular changes at serial time points following total lung irradiation. Materials and Methods: Adult, female C57BL/6J mice received 20 Gy irradiation to both lungs and subgroups of mice were examined at days 0, 7, 14, 80, 100, 120, or at the time of death, for immunohistochemically detectable E-selectin, P-selectin, VCAM-1, and ICAM-1. The phenotype of cells with macrophage (F458 and MAC-1+), endothelial cells (vWf and endothelin), or fibroblast positivity (cytokeratin) was correlated with detectable upregulation of each adhesion molecule. At each time point, the mice were pulsed with BrdU one hour prior to sacrifice, and immunohistochemically detectable uptake of BrdU in dividing cells was correlated to areas of upregulated adhesion molecules and proliferation. In addition, female C57BL/6J, green fluorescent protein-negative (GFP-) recipient mice were engrafted prior to pulmonary irradiation with GFP+ male bone marrow, and donor marrow-derived macrophages and fibroblasts in the lungs were scored at the time of death. The possible bone marrow origin of the fibrotic lesions was quantitated in other 20 Gy total lung irradiated mice that received 1x105 cells from a clonal GFP+ bone marrow stromal cell line at 60 or 100 days after 20 Gy irradiation. Results: After the acute reaction (14 days after irradiation) no detectable increase in adhesion molecules was observed until day 80, when VCAM-1 and ICAM-1 levels were predominantly increased in pulmonary endothelial cells, and also in lung parenchymal cells. The first significant BrdU uptake was observed at day 100 and correlated with fibrotic lesions, primarily in fibroblasts, and also in F480+ macrophages. A significant number of pulmonary macrophages detected at day 100 after 20 Gy irradiation were of bone marrow origin. Fibrotic lesions detected between days 100 and 120 were comprised of GFP+ bone marrow-derived fibroblasts. Mice receiving intravenous injection of 1x105 cells of the clonal GFP+ bone marrow stromal cell line at day 100 after 20 Gy irradiation demonstrated significant adhesion and proliferation of circulating fibroblast progenitor cells in their lungs, compared to nonirradiated mice. Conclusion: Late pulmonary irradiation fibrosis (organizing alveolitis) in the C57BL/6J mouse model is associated with a detectable increase in VCAM-1 and ICAM-1 in endothelial and pulmonary parenchymal cells prior to detectable fibroblast proliferation by BrdU labelling. Bone marrow-derived macrophages and fibroblasts comprise a significant fraction of fibrotic/organizing alveolitis lesions in the lungs of C57BL/6J mice after day 100. Demonstration of the bone marrow origin of cells comprising fibrotic lesions in the lungs of C57BL/6J mice supports a dynamic model of pulmonary fibrosis, and suggests new targets for therapeutic intervention by preventing bone marrow stromal cell homing to the irradiated lung.