Interleukin-3 (IL-3) (multipotential colony-stimulating factor [multi- CSF]) is an important regulator of hemopoiesis. We recently isolated a cDNA clone of this gene and describe in this manuscript the biologic properties of the expressed gene product. An SV40 expression vector carrying cDNA encoding murine interleukin-3 was constructed so that expression of the IL-3 gene was placed under the control of the SV40 early promoter. When the expression vector was transfected to COS-1 monkey cells, IL-3 activity was secreted into the medium, reaching maximal levels 72 hours after transfection. The IL-3 produced by the COS-1 cells was partially purified using diethylaminoethyl Sephacel and phenyl-Sepharose, and its chromatographic properties were the same as IL-3 produced by the WEHI-3 cell line. The biologic activities of the “expressed” IL-3 include the “induction”of 20-alpha-hydroxysteroid dehydrogenase (20-alpha-SDH) in splenic lymphocytes from nu/nu mice, proliferative activity for 32D cl-23 and FDC-P1 cell lines, and colony- stimulating activity for granulocyte-macrophage, eosinophil, megakaryocyte, natural killer-like, erythroid, and multipotential colony-forming cells from murine fetal liver and adult bone marrow.

The optimum marrow ablative regimen for preparing recipients of bone marrow transplantation (BMT) has not been established. gamma- Irradiation, but not busulfan, produces a characteristic microvascular injury pattern which results in depressed capacity of normal lymphocytes to localize into the lymph nodes of syngeneic murine BMT recipients. Since peripheral lymph nodes are important sites for initiation and amplification of immune responses, the preparative regimen might delay recovery of regionally compartmentalized immune functions after BMT. We evaluated the effects of busulfan and gamma- irradiation on the phenotypic and functional reconstitution of helper T- cell function within the peripheral lymph nodes of BMT recipients. Both marrow ablative regimens caused a protracted delay in regeneration of peripheral lymph node CD4+ T cells. Specific helper T-cell functions, such as contact hypersensitivity and alloantigen responses, remained significantly depressed in the lymph nodes of irradiated mice for prolonged periods (up to 60 weeks). These responses recovered more rapidly in busulfan-treated BMT recipients. In contrast, the capacity of peripheral lymph node T cells to provide “help” for antigen-specific immunoglobulin production was only transiently depressed by either preparative regimen. Our experiments confirm the hypothesis that the marrow ablative regimen, particularly gamma-irradiation, may contribute to the period of immunodeficiency which follows BMT. The pattern of immune recovery observed suggests that preparative total body irradiation (TBI) may selectively depress the regional recovery of the TH1 [interleukin-2 (IL-2) and gamma-interferon (gamma-IFN) secreting] lymphocyte subset.

The enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) has been proposed as a T lymphocyte marker that is specifically induced by interleukin 3. We have examined the expression of 20 alpha SDH and its relationship to interleukin 3 responsiveness in other hemopoietic lineages. The enzyme is expressed at high levels in the bone marrow of athymic nu/nu mice, but only at low levels in nu/nu spleen and normal adult bone marrow. 20 alpha SDH is induced in nu/nu spleen and in normal fetal liver cells by granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as by interleukin 3. In longer term cultures of fetal liver and adult marrow, macrophage colony-stimulating factor (M-CSF) also induces the enzyme, which is expressed in proliferating adherent macrophages. The high levels of 20 alpha SDH in purified bone marrow-derived macrophages are maintained by M-CSF, GM-CSF, or interleukin 3. Expression of 20 alpha SDH in these cells is associated with resistance to growth inhibition by progesterone. Additional evidence against the restriction of 20 alpha SDH to T lymphocytes is found in its presence in peritoneal macrophages, myelomonocytic and macrophage-like cell lines, and the L929 fibrosarcoma line. We conclude that 20 alpha SDH is a normal enzyme of proliferating hemopoietic cells irrespective of their lineage or growth factor responsiveness.