Various techniques are available for distinguishing donor from host cells evaluating the efficacy of conditioning regimen for experimental bone marrow transplantation (BMT). Techniques include the use of extracellular immunological markers, such as Ly5 (CD45), and intracellular biochemical markers, such as glucose-phosphate-isomerase (Gpi). Because Ly5 is an extracellular protein, the disparity between donor (Ly5.1) and host (Ly5.2) antigens may induce a weak immune response whereas with Gpi, no immune response is expected. This difference may be of particular concern in experimental transplantation approaches that use minimal conditioning such as low-dose total body irradiation (TBI). Such mild conditioning may not induce the immunosuppression required to overcome host rejection of Ly5 disparate cells.To ). However, when higher TBI or BMC doses were used, similar engraftment levels were found, suggesting sufficient immune suppression to allow equal engraftment of both sources of BM.These data suggest that even a minor phenotypic disparity between donor and host, such as Ly5, may necessitate high-dose TBI to prevent rejection. The combination of low-dose TBI or other nonmyeloablative conditioning strategies with small numbers of BMC may lead to reduced engraftment when extracellular immunological markers such as Ly5 are used for transplantation studies. Therefore, small immunological differences must be considered when using the Ly5 marker for engraftment.

It has been suggested that mature neutrophils may play an essential role in the cascade of events leading to egress of stem cells from the bone marrow to the peripheral blood. To investigate further the role of mature neutrophils and of reactive oxygen intermediates (ROIs), known to be involved in the signal transduction of neutrophils, we used mice deficient in respiratory burst, and thus the production of ROIs, to study the involvement of this activation pathway in stem cell mobilization. B6 mice with chronic granulomatous disease (CGD) received either cyclophosphamide (200 mg/kg) on day 1 and granulocyte colony‐stimulating factor (G‐CSF) (250 µg/kg/d) on days 3–6 or a single dose of interleukin 8 (IL‐8; 30 µg/mouse) as a mobilization regimen. On day 7, the number of stem and progenitor cells in blood and bone marrow was compared with control B6 animals (with intact respiratory burst). White blood cell counts, bone marrow cellularity and the frequency of granulocyte–macrophage colony‐forming cells (GM‐CFC), and cobblestone area‐forming cells (CAFC) on days 7 (CAFC‐7) and 28 (CAFC‐28) were determined. After cyclophosphamide and G‐CSF (CY + G), both mouse strains showed considerable mobilization of CAFC‐7 and CFU‐GM to the blood. Normal mice showed up to a 1905‐fold increase in progenitors per ml blood, whereas CGD mice showed up to a 264‐fold increase in blood progenitors. IL‐8 also induced mobilization in both mouse strains. In addition to progenitors, primitive stem cells measured as CAFC‐28 and as CAFC at day 35 were also mobilized by both mobilization protocols in normal as well as in CGD mice. In conclusion, respiratory burst and the subsequent signal transduction pathway do not appear to be required for mobilization of stem cells. Accordingly, neutrophils either are not involved in stem cell mobilization or other signalling pathways within neutrophils must exist that lead to the release of factors which activate stem cell egress from the bone marrow.