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In order to facilitate the identification of human haemopoietic growth factors (HGFs), in complex conditioned media from limited cell sources, we have developed a method for the rapid assay of HGFs using bone marrow stem cells (BMSCs) obtained by Percoll density gradient enrichment of non-adherent cells from long-term human bone marrow culture. These BMSCs provide the basis for a 72 h assay where proliferation is triggered by the presence of GM-CSF, G-CSF and IL-3. The assay is up to 1000 times more sensitive than the conventional colony assay in detecting the presence of HGFs. We also describe methods for the rapid separation and identification of HGFs using fast protein liquid chromatography (FPLC) with phenyl-Superose and Mono Q columns. This enables individual HGFs present in complex conditioned media to be rapidly identified from the elution profile of HGF activity determined in the BMSC assay.

The rate of mobilisation of macrophages from marrow is an important determinant of the outcome of many pathological lesions. Studies of BMDM have provided insight into the complex feedback regulation that may control macrophage production. In addition BMDM possess several properties in common with transformed cells of other tissue origins and an understanding of their growth regulation may provide an insight into the mechanism of malignant transformation.

Spleen cells from mice immunized with vaccinia or influenza viruses were mixed with spleen cells from mice immunized by dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) and transferred into irradiated syngeneic recipient mice which were subsequently restimulated with trinitrophyenylates (TNP) or unmodified viruses. One week later, spleens were removed and assayed for indirect anti-DNP plaque-forming cells (PFC). When spleen cells from both influenza and vaccinia primed mice were restimulated with the haptenated form of the virus used for the initial immunization there was an enhanced PFC response compared to that seen with spleen cells from unprimed mice.

It has been recently demonstrated that conditioned media from Con A-stimulated splenocyte cultures contain a novel lymphokine termed IL 3. IL 3 is characterized by its ability to induce 20-alpha-hydroxysteroid dehydrogenase in spleen cells from young nu/nu mice. In search of a convenient source for biochemical and in vivo studies of this lymphokine, a number of established murine cell lines were screened for the constitutive and induced production of Il 3. It was found that WEHI-3 cells, originally designated as a myelomonocyte cell line, produced high levels of IL 3 constitutively. Added mitogen and/or phorbol acetate did not enhance the production of IL 3. Production of IL 3 varied among various sublines of WEHI-3. IL 3 purified from WEHI-3-conditioned media has biochemical and biologic characteristics identical to those obtained from Con A-conditioned media. WEHI-3 conditioned media, however, generally contained 100-fold higher levels of IL 3 than conditioned media from Con A-stimulated splenic lymphocytes.

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.

IL-3 is a recently described lymphokine that induces the expression of the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in an early T-cell precursor. We demonstrate that purified IL-3 an be used to establish continuous cultures of a discrete subpopulation of T cells with virtually 100% efficiency from normal, unstimulated splenic lymphocyte populations. Once established over a period of approximately 4-6 weeks, the cultures can be readily cloned either in soft agar or by limiting dilution. All of the established lines are Lyt1+, 2- la-, lg-, Tdt-, 20 alpha SDH+, a phenotype characteristic of helper T cells; they are therefore distinct from continuous cultures of T cells established with IL-2. although initiation of these cell lines was absolutely dependent on IL-3, once established all of the cell lines were independent of exogenously added lymphokines for their growth in vitro. However, all of the cells lines were found to constitutively produce IL-3 at high levels. None of the cell lines constitutively produced lL-2, but could be readily induced to produce this lymphokine by treatment with phorbol-myristic acetate. The ability to produce lL-3 and lL-2 is a further indication that all the cell lines are helper T cells. The possible mechanisms by which lL-3 allows the specific differentiation and/or amplification of T cells of helper phenotype in tissue culture are discussed.

The clonal growth in nutrient agar at low cell densities of high-proliferative potential colony-forming cells (HPP-CFC) of bone marrow obtained from mice treated 2 days earlier with 5-fluorouracil (FU) (FU2dBM) has been shown to require a combination of three growth factors, interleukin 1 (IL-1), interleukin 3 (IL-3), and macrophage colony-stimulating factor (CSF-1). These HPP-CFC have been enriched 140-fold from FU2dBM by fluorescence-activated cell sorting of 7/4-, B220-, and L3T4-negative cells. The mean of the plating efficiencies of these enriched populations was 4.4% and no growth was observed when the factors were used singly. Similarly, enrichments of 16-fold were obtained from FU2dBM using immunomagnetic Dynabeads with anti-7/4 plus anti-B220 (meaning plating efficiency 0.5%). The further additions of human granulocyte CSF or mouse granulocyte-macrophage CSF or both to IL-1 plus IL-3 plus CSF-1 did not increase HPP-CFC colony formation, but both augmented the small colony formation with IL-1 plus IL-3, IL-3 plus CSF-1, or IL-1 plus CSF-1.

Genomic clones carrying the rat interleukin-3 (IL-3) gene have been isolated and the nucleotide sequence of the gene determined. Alignment of this sequence with that of the mouse IL-3 gene has allowed the structure of the rat IL-3 gene to be deduced. The intron-exon boundaries are conserved and extensive nucleotide homology (approx 90%) is present in the 5' flanking region and the portion of the gene coding for the signal peptide. Several proposed regulatory sequences are conserved and an analogous element to the tandem repeat in intron 2 of the mouse gene is also present. The predicted amino acid sequence for mature rat IL-3 shows surprisingly low homology (54%) with its murine counterpart, although all four cysteine residues are conserved. The rat IL-3 gene was expressed in monkey COS-1 cells and colony assays established that rat IL-3 is a multi-lineage haemopoietic growth regulator. There was little cross-reactivity of the respective IL-3 species on mouse and rat bone marrow cells suggesting that rat IL-3, in concert with its receptor, has evolved significantly away from the mouse IL-3/receptor system.

The inflammatory response in mouse brain and the elimination of virus from the brain following intracerebral inoculation of a group A arbovirus has been shown to be T-cell dependent. Virus persists in the brain of T-cell depleted mice, and inflammation is depressed. Virus persists in the brain of T-cell depleted mice, and inflammation is depressed. Inflammation is restored by transfer of immune cells but not by immune serum, and the transferred cells are effective in reducing virus titres in the absence of circulating antibody. Transferred antibody is not efficient in protecting infected mice. The cells that restore inflammation can come from mice immunized with a group A arbovirus of the same subtype but not from mice immunized with more distantly related viruses. Macrophages may be important effector cells working in collaboration with T cells.

The cDNA sequence for murine interleukin-3, one of the colony stimulating factors that regulate haematopoiesis, codes for a polypeptide of 166 amino acids including a putative signal peptide. The predicted amino acid sequence indicates that formation of mature interleukin-3 involves proteolytic removal of not only the signal peptide but additional amino-terminal amino acids.

Interleukin-3 (IL-3) can promote the proliferation of certain classes of lymphocytes distinct from those that are dependent on interleukin-2 (IL-2) for growth. Culture conditions for its production are identical to those required for IL-2 and in both cases the producer cell appears to be Thy 1.2+, Lyt1+, Lyt2- However, unlike the IL-2 responder cells, the cells that proliferate in IL-3 are generally Lyt2-. Here we have measured the natural cytotoxic (NC) activity as well as natural killer (NK) cell activity of the IL-3-dependent cells. Both of these activities are part of a repertoire of spontaneous cytotoxic functions found in mice that might serve in early defence against infectious agents or immunosurveillance against tumours in vivo. We report a new finding that IL-3 selectively maintains NC cells but not NK cells in culture. This is in contrast to the known requirement for IL-2 in NK cell growth. This provides a means of isolating NC cells from NK cells in the mouse as an initial step in the study of the relative contribution of these two cell types in tumour immunity.

Interleukin-3 (multi-CSF) is a multilineage haematopoietic growth regulator that initiates the proliferation and differentiation of multipotential stem cells. Complementary DNA clones encoding interleukin-3 (IL-3) have recently been isolated and the structure of the IL-3 gene determined. IL-3 is produced by T lymphocytes or T lymphomas only after stimulation with antigens, mitogens or chemical activators such as phorbol esters. The myelomonocytic leukaemia line WEHI-3B also produces IL-3 but its production is constitutive and the WEHI-3B cells do not appear to produce significant levels of any of the other lymphokines normally secreted by T lymphocytes after stimulation. It has been proposed that the genetic change leading to the constitutive expression of IL-3 may have been a key event in the development of this leukaemia. We report here that the constitutive synthesis of IL-3 by the WEHI-3B cell line is due to the insertion of an endogenous retrovirus-like element close to the 5' end of the gene. The insertion, an intracisternal A particle (IAP) genome, is positioned with its 5' long terminal repeat (LTR) close to the promoter region of the IL-3 gene, resulting in constitutive synthesis of IL-3.