When investigating the biological effects of ionizing radiation on the haemopoietic system, a confounding problem lies in possible differences between the biological effects of sparsely ionizing, low linear energy transfer radiation such as X-, beta- or gamma-rays, and densely ionizing, high linear energy transfer radiation such as alpha-particles. To address this problem we have developed novel techniques for studying haemopoietic cells irradiated with environmentally relevant doses of alpha-particles from a plutonium-238 source. Using a clonogenic culture system, cytogenetic aberrations in individual colonies of haemopoietic cells derived from irradiated stem cells have been studied. Exposure to alpha-particles (but not X-rays) produced a high frequency of non-clonal aberrations in the clonal descendants, compatible with alpha-emitters inducing lesions in stem cells that result in the transmission of chromosomal instability to their progeny. Such unexpected instability may have important implications for radiation leukaemogenesis.

Haemopoietic tissues exposed to ionizing radiation are shown to exhibit increased macrophage activation, de®ned by ultrastructural characteristics and increased lysosomal and nitric oxide synthase enzyme activities. Macrophage activation post-irradiation was also associated with enhanced respiratory burst activities and an unexpected neutrophil in®ltration. Examination of p53-null mice demonstrated that macrophage activation and neutrophil in®ltration were not direct e ects of irradiation, but were a consequence of the recognition and clearance of radiation-induced apoptotic cells. Increased phagocytic cell activity was maintained after apoptotic bodies had been removed. These ®ndings demonstrate that, contrary to expectation, recognition and clearance of apoptotic cells after exposure to radiation produces both a persistent macrophage activation and an in¯am-matory-type response. We also demonstrate a complexity of macrophage activation following radiation that is genotype dependent, indicating that the in vivo macrophage responses to radiation damage are genetically modi®ed processes. These short-term responses of macrophages to radiation-induced apoptosis and their genetic modi®cation are likely to be important determinants of the longer-term consequences of radiation exposure. Furthermore, in addition to any e ects attributable to immediate radiation-induced damage, our ®ndings provide a mechanism for the production of damage via a`bystander' e ect which may contribute to radiation-induced genomic instability and leukaemogenesis. Oncogene (2001) 20, 7085 ± 7095.

The haemopoietic system has three main compartments: multi-potential stem cells, intermediate stage progenitor cells and mature cells. The availability of simple reproducible culture systems has made possible the characterization and purification of regulators of the progenitor cells, including colony-stimulating factors and interleukins. In contrast, our knowledge of the regulators involved in the control of stem cell proliferation is limited. The steady-state quiescent status of the haemopoietic stem cell compartment is thought to be controlled by locally acting regulatory elements present in the stromal microenvironment, but their purification has been hampered by the lack of suitable culture systems. We have recently developed a novel in vitro colony assay that detects a primitive cell (CFU-A) which has similar proliferative characteristics, in normal and regenerating bone marrow, to the CFU-S (haemopoietic stem cells, as defined by the spleen colony assay) and which responds to CFU-S-specific proliferation regulators. We have now used this assay to purify to homogeneity a macrophage-derived reversible inhibitor of haemopoietic stem cell proliferation (stem cell inhibitor, SCI). Antibody inhibition and sequence data indicate that SCI is identical to a previously described cytokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), and that SCI/MIP-1 alpha is functionally and antigenically identical to the CFU-S inhibitory activity obtained from primary cultures of normal bone marrow cells. The biological activities of SCI/MIP-1 alpha suggest that it is a primary negative regulator of stem cell proliferation and that it has important therapeutic applications in protecting haemopoietic stem cells from damage during cytotoxic therapies for cancer.

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