Effects of radiation and 4-hydroperoxycyclophosphamide on production of G- and GM-CSF by stromal cells

The effects of in vitro radiation and exposure to 4-hydroperoxycyclophosphamide (4-HC) on the production of G- and GM-CSF by different components of the human hematopoietic microenvironment are described. The marrow microenvironment is composed of fibroblasts, endothelial cells, macrophages, and adipocytes. To study the effects of radiation/4-HC on colony-stimulating factor (CSF) production by stromal cells, confluent layers of umbilical cord endothelial cells (EC), marrow fibroblasts (MF), and heterogeneous adherent layers (HAL) derived from long-term marrow cultures were established. These layers were exposed to radiation up to 3000 cGy and/or 100 μmol/ml of 4-HC and subsequently stimulated with IL-1β on day 0, 7, or 14 after radiation/4-HC. Following IL-1 exposure conditioned medium (CM) was collected and G- and GM-CSF levels were measured by ELISA and their ability to support colony formation was assessed. G- and GM-CSF levels after exposure to 4-HC and radiation were 12460 ± 172 pg/ml and 2268 ± 160 pg/ml for EC, 2214 ± 94 pg/ml and 263 ± pg/ml for MF, and 3168 ± 316 pg/ml and 356 ± 34 pg/ml for HALs, respectively. For each cell group there was no significant difference between levels obtained without exposure and levels after exposure to 4-HC and/or radiation (p > 0.6). Comparison of levels obtained from different cell groups showed significant differences with EC media being the highest (p < 0.0001). To test the activity of these measured factors, CM of different sources was used in colony assays of CD 34+ cord blood progenitors. Day 7 and 14 colonies per 10³/CD 34+ cells developing in the presence of different CM collection after radiation and/or 4-HC exposure, were scored. CM collected after exposure to 4-HC and radiation supported 39 ± 3 and 64 ± 6 day 7 and 14 CFU-GM colonies, respectively, for EC, 16 ± 2 and 37 ± 2 for MF, and 23 ± 6 and 47 ± 7 for HALs. These CM also supported the development of BFU-E. Similar colony frequency was obtained from CM obtained after 4-HC or radiation alone. Addition of anti-G-CSF and anti-GM-CSF reversed the growth of day 7 and day 14 colonies, respectively. Our data indicate that radiation and/or 4-HC do not impair stromal cell capacity to produce G- and GM-CSF.