Biologic properties of homogeneous interleukin 3. I. Demonstration of WEHI-3 growth factor activity, mast cell growth factor activity, p cell-stimulating factor activity, colony-stimulating factor activity, and histamine-producing cell-stimulating factor activity

Interleukin 3 (IL 3) was initially defined as a factor in conditioned media from concanavalin A-stimulated lymphocytes (Con A CM) that induces the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) in cultures of nu/nu splenic lymphocytes. To determine the spectrum of additional "biologic" activities, IL 3 was purified to homogeneity and its properties were assessed. The protein preparation was judged to be homogeneous IL 3 by the following criteria: 1) elution of a peak of IL 3 with a constant specific activity in the last step of purification, 2) presence of a single protein by SDS-PAGE analysis, 3) receptor-binding activity against IL 3-dependent cell lines, 4) a specific activity of congruent to 0.2 ng/ml required for 50% of maximal biologic activity, and 5) the presence of a single amino terminal sequence. With the use of this preparation of IL 3, the dose-response curves for 20 alpha SDH induction were identical or similar to the dose-response curves for the activities of 1) WEHI-3 growth factor, 2) mast cell growth factor, 3) P cell-stimulating factor, and 4) histamine-producing cell-stimulating factor. In addition, homogeneous IL 3 had colony-stimulating factor activity, although only approximately 2% of the total CSF activity found in Con A CM was associated with IL 3. The major peak of CSF activity could be resolved from IL 3 by DEAE column chromatography and lacked many of the biologic activities associated with IL 3.     

Interleukins 2 and 3 regulate the in vitro proliferation of two distinguishable populations of 20-alpha-hydroxysteroid dehydrogenase-positive cells

The ability of interleukin 2 (IL 2), interleukin 3 (IL 3), and granulocyte/macrophage colony-stimulating factor (GM-CSF) to induce the proliferation of cells from thymus, spleen, or bone marrow was examined and compared with their ability to induce expression of the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH). In the thymus, the peanut agglutinin agglutinated cells (PNA+) lacked 20 alpha SDH and showed no detectable response to IL 2, IL 3, or GM-CSF in either proliferation or induction of 20 alpha SDH. In contrast, the PNA nonagglutinated (PNA-) subpopulation expressed 20 alpha SDH and proliferated in response to Con A and/or IL 2. The responding cells that could be expanded in vitro with IL 2 expressed high levels of 20 alpha SDH. Neither IL 3 nor GM-CSF in the presence or absence of Con A had a demonstrable effect on the PNA- population. In cultures of bone marrow cells, both IL 3 and GM-CSF induced proliferation, whereas IL 2 had no effect on proliferation in the presence or absence of Con A. Thy-1-depleted bone marrow cells, expanded in tissue culture with IL3, contained cells that co-expressed Thy-1 and 20 alpha SDH. In contrast, cells proliferating in vitro to GM-CSF did not expressed Thy-1 or 20 alpha SDH. In cultures of normal splenic lymphocytes, two populations of cells capable of expressing 20 alpha SDH were detected. One population could be expanded in vitro with IL 2 and Con A, whereas the second was responsive to IL 3. In spleens from athymic mice, only the latter cells were detected. These results demonstrate that IL 3 and IL 2 responsiveness distinguishes two populations of 20 alpha SDH cells. The relevance of these observations to the possible relationship of IL 3 and IL 2 in T cell differentiation is discussed.     

Constitutive production by the WEHI-3 cell line of B cell growth and differentiation factor that co-purifies with interleukin 1

The myelomonocytic cell line WEHI-3 produces constitutively a factor that affects the growth and differentiation of murine B cells in culture. This cell line also secretes colony-stimulating factors (CSF), interleukin 1 (IL 1) but not interleukin 2. Sequential purification through AcA54 gel filtration, DEAE-Sephacel ion exchange chromatography, and buffer electrofocussing clearly resolved the B cell growth and differentiation factor (BGDF) from the CSF activities but failed to separate BGDF from IL 1. The WEHI-3-derived material responsible for BGDF/IL 1 activity, however, exhibited different behavior on DEAE chromatography (elution at 175 mM NaCl) to that reported for IL 1 from the P388D1 cell line (elution at 50 mM NaCl). B cell growth and differentiation could be induced by WEHI-3 BGDF/IL 1 in cultures of normal spleen cells depleted of T cells and adherent cells but not in cultures of spleen cells from B cell-deficient CBA/N mice, even though thymocytes from such mice displayed a normal response to IL 1. Significant B cell proliferation induced by BGDF/IL 1 was apparent only in the presence of submitogenic concentrations of anti-mouse IgM antibodies, but under these conditions few antibody-forming cells (AFC) were generated. In contrast, B cell differentiation to AFC occurred in the presence of the factor alone, and this response was inhibited by anti-IgM. Thus there appeared to be a reciprocal relationship between B cell proliferation and differentiation induced by BGDF/IL 1. The significance of these results is discussed in the light of other recent studies of BGDF.     

Astrocyte-derived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages

Astrocytes have been shown to release an interleukin 3 (IL 3)-like factor that induces the expression of 20-alpha-hydroxysteroid-dehydrogenase (20-alpha SDH) in nu/nu spleen cells, and the proliferation of the IL 3-dependent cell line 32DCL. We have investigated whether astrocyte-derived IL 3 supports growth of macrophages and their representatives in the brain, the microglia cells. Evidence for intercellular communication between murine astrocytes and macrophages became already detectable in co-culture experiments: astrocytes activated with endotoxin resulted in an increased growth of peritoneal macrophages on the astrocyte monolayer. Biochemical analysis of supernatants of activated astrocytes revealed that the IL 3-like factor that stimulated 32DCL cells and the expression of 20 alpha SDH also served as a growth factor for cultured peritoneal macrophages. The same results were obtained by using microglia cells isolated from primary brain cell cultures of newborn mice, which are characterized by their positive reaction for macrophage markers such as Mac-1 and nonspecific esterase. If secreted by reactive astrocytes in vivo, the IL 3-like factor may contribute to the accumulation of macrophages and microglia cells detected in brain lesions of patients with multiple sclerosis.     

Establishment of continuous cultures of thy1.2+, Lyt1+, 2-T cells with purified interleukin 3

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.     

Interleukin 2-dependent natural killer (NK) cell lines from patients with primary T cell immunodeficiencies

Bulk cultured cell lines with natural killer (NK) activity were derived by in vitro culture with interleukin 2-containing conditioned medium (IL 2-CM) of peripheral blood mononuclear leukocytes (PBL) from patients with primary T cell deficiencies. Lines were developed from three patients with severe combined immunodeficiency (SCID) and one patient with Nezelof's syndrome and contained several populations of cells with distinct phenotypes. All lines contained a cell population expressing the Leu-5 (50K) (sheep red blood cell receptor), 3A1 (40K), and OKT10 antigens, but lacking the pan T cell antigens Leu-1 (67K) and Leu-4 (19K) as well as the markers of T cell subsets Leu-2a (32K) and Leu-3a (56K). These cells failed to express the Leu-7 antigen and only weakly expressed OKM1. In addition, one line contained a population of Leu-5+, 3A1+, OKT10+, Leu-2a+, Leu 1-, and Leu 4- cells. Three of the lines also contained populations with classic T cell (Leu-1 and-Leu 4+) phenotypes. The lines were enriched in NK activity compared with the PBL from which they were derived. Their growth was strictly dependent on IL 2-CM. Highly purified IL 2, lacking any other detectable protein contaminants or lymphokine activities, was capable of supporting the growth of the Leu-5+, 3A1+ "null" cell populations from these lines without alteration in their functional activity or phenotype. Thus, studies of in vitro expanded cell lines from patients with severe disorders of T cell function and thymic involution indicate that this "null" cell population does not require thymic maturation to develop its effector function. This "null" cell population can be maintained in vitro in the presence of IL 2. This finding is analogous to the data obtained from study of NK cells in athymic (nude) mice.     

Self-renewal of factor-dependent hemopoietic progenitor cell-lines derived from long-term bone marrow cultures demonstrates significant mouse strain genotypic variation

Long-term bone marrow cultures established from C57Ks/J mice have been shown to spontaneously release endogenous ecotropic RNA type-C virus (retrovirus). C57Ks/J marrow cultures produced granulocyte-macrophage progenitor cells (GM-CFUc) and immature and mature granulocytes for over 45 weeks. In contrast, NIH Swiss mouse marrow cultures failed to release detectable ecotropic virus and generated GM-CFUc and granulocytes for 25–35 weeks and established WEHI-3 conditioned medium (CM) dependent cell lines in vitro and did not establish permanent cell lines. To determine whether viral and/or cellular genes regulated the longevity of C57Ks/J marrow cultures, groups of cultures were established from the marrow of (NIH-Swiss × C57Ks/J) F1 hybrid, F2 hybrid, and (NIH Swiss × C57Ks/J) X NIH Swiss backcross generations. Release of endogenous ecotropic virus was measured weekly in each culture as was the duration of production of immature granulocytic cells and GM-CFUc over a 58-week period. The results demonstrated a complex pattern of inheritance of longevity of long-term in vitro hemopoiesis. Increased longevity did not absolutely correlate with detectable replication of the C57Ks/J N-tropic virus.     

Interleukin 3-dependent hematopoietic progenitor cell lines

Several biological phenotypes of growth factor-dependent cell lines have been described in recent years, including those with T lymphocyte, neutrophil granulocyte, basophil/mast cell, B lymphocyte, and multipotential stem cell properties. The growth factors for each cell lineage are a subject of intense study. Continuous mouse bone marrow cultures infected with RNA type C viruses (retroviruses) produce nonadherent hematopoietic cells over a longer duration than control cultures. Marrow cultures derived from strains with spontaneously induced ecotropic endogenous retrovirus demonstrate a greater longevity than those from strains with no replicating virus. Cultures infected with murine leukemia virus also generate a greater number, compared with controls, of cloned permanent suspension cell lines dependent for growth on a 41,000-dalton glycoprotein (interleukin 3 [IL 3]). Some are multipotential with capacity for differentiation to erythroid, neutrophil, eosinophil, and basophil/mast cell types. Other cloned IL 3-dependent cell lines are committed to a single pathway. Studies with Friend spleen focus-forming virus indicate that the first effect in the marrow culture is mediated through a subset of adherent hematopoietic stem cells. Bone marrow culture-derived IL 3-dependent cell lines provide a model with which to study the role of viral genes in the control of differentiation and self-renewal capacity of hematopoietic stem cells.     

Astrocytes of the brain synthesize interleukin 3-like factors

Interleukin 3 (IL 3) is produced by T lymphocytes and T cell lines, as well as by a myelomonocytic cell line (WEHI-3), and it activates lymphocytes and mast cells, as well as macrophages. Recently we have demonstrated that astrocytes act as immune accessory cells through the secretion of interleukin 1 and the presentation of antigens to T lymphocytes. Here we show that cultured astrocytes from newborn mice release a 30,000 m.w. factor that induces the expression of 20-alpha-hydroxysteroid dehydrogenase in nu/nu spleen cells and the proliferation of the IL 3-dependent cell line 32DCL. An analogous biological activity was detected in supernatant of cultured rat C6 glioma cells. Production of IL 3-like factors by astrocytes of the central nervous system may be essential for development and maintenance of hemo and lymphopoietic cells within inflammatory brain lesions.     

TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL 2 receptor on a natural killer-like cell line (YT cells)

A continuous cell line (YT cells) with inducible receptor for T cell growth factor (TCGF)/interleukin 2 (IL 2) was established from a 15-yr-old boy with acute lymphoblastic lymphoma and thymoma. YT cells were tetraploid, having 4q+ chromosomal markers, and proliferated continuously in vitro without conditioned medium (CM) or IL 2. They were weakly positive for OKT9, OKT11, and Tac antigen (Ag), a determinant closely associated with the receptor for IL 2 (IL 2-R), and were negative for OKT1, OKT3, OKT4, and OKT8 Ag. YT cells also expressed HNK-1 Ag and Fc receptors for IgG, which are expressed on natural killer (NK) cells. They retained a killing activity against human cell lines, including K562 (myeloid), T, and B cell lines. Unlike Tac Ag/IL 2-R(+) cell lines derived from adult T cell leukemia (ATL), YT cells were negative for HTLV, as proved by Southern blotting with cDNA for viral DNA. The expression of Tac Ag was markedly enhanced in 18 hr, when YT cells were incubated with CM from PHA-stimulated peripheral blood leukocytes (PBL) or spleen cells, as determined by immunofluorescence by using flow cytometry and binding assay with 125I-anti-Tac antibody (Ab). The binding study with 125I-labeled recombinant IL 2 showed 3.2 X 10(4) IL 2 receptor sites on YT cells precultured with CM. PHA-P and Con A neither agglutinate nor enhance the expression of IL 2-R/Tac antigen on these non-T cell line cells. Furthermore, neither recombinant IL 2 nor gamma-interferon could induce IL 2-R on YT cells, suggesting the presence of a unique IL 2-R inducing factor in PBL or spleen CM. Unlike Tac Ag on HTLV(+), ATL-derived cell lines (Hut-102, MT-1, ATL-2), the expression of Tac Ag on YT cells was down-regulated by anti-Tac Ab. The induction of Tac Ag/IL 2-R on YT cells seemed specific, because the enhancement of Tac Ag expression was not associated with that of Ia Ag and T9/transferrin receptor.     

Long-term in vitro culture of murine mast cells. III. Discrimination of mast cells growth factor and granulocyte-CSF

Long-term in vitro growth of murine mast cells was dependent on the presence of a mast cell growth factor (MCGF) present in media conditioned by mitogen-activated splenic leukocytes or by various murine leukemic cell lines. MCGF shared a number of properties with granulocyte colony-stimulating factor (G-CSF). Both factors were present in media conditioned by the myelomonocytic leukemic WEHI-3 and the T cell lymphoma, LBRM-33 cell lines. They were relatively sensitive to trypsin treatment, and were resistant to boiling temperature. NZB mice that failed to respond to WEHI-3-derived G-CSF also failed to respond to MCGF. MCGF differed from G-CSF, however, in sensitivity to neuraminidase and lactoferrin, an inhibitor of macrophage CSF production, suppressed G-CSF production by WEHI-3 cells without affecting MCGF production. Furthermore, peritoneal cells produced G-CSF but not MCGF when stimulated with lipopolysaccharide. In vitro production of MCGF by normal spleen cells required the presence of T lymphocytes and is relatively macrophage-independent. The role of T cells in the maturation and growth of mast cells and the physiologic function of MCGF are discussed.     

Two-factor requirement for murine megakaryocyte colony formation

WEHI-3 cell-conditioned medium with the capacity to stimulate megakaryocyte colony formation was separated by Sephadex G-150 column chromatography. The development of colonies containing megakaryocytes was observed only when mixing experiments were performed. Individual fractions did not support megakaryocyte colony growth. The two factors in WEHI-3 CM required for megakaryocyte colony growth had apparent average molecular weights of 35,000 daltons (megakaryocyte CSF) and 100,000 daltons (megakaryocyte potentiator). The results were confirmed in serum-free conditions in which colonies were directly identified in the cultures by acetylcholinesterase staining. Two growth factors may be necessary for the genesis of megakaryocytic colonies.     

Stimulation of interleukin 1 and 3 production by retinoic acid in vitro.

Retinoic acid and other retinoids stimulate or inhibit a number of immune responses, but their mechanism of action on immune cells is not fully understood. However, retinoids have been shown to inhibit interferon production, so they could act by influencing the production of lymphokines. Hence we have studied the effect of retinoic acid on the production of interleukins (ILs) 1 and 3 in vitro. Models for the production of ILs were the murine macrophage cell line P388D1 and human peripheral blood mononuclear cells for IL 1 and the murine WEHI-3 cell line for IL 3. Retinoic acid stimulated IL 1 release by P388D1 cells in a dose-related fashion, starting at 10(-9) M and maximally at 10(-8)-10(-6) M. With peripheral blood mononuclear cells a maximal stimulation of IL 1 release was observed with 10(-7) M-retinoic acid. IL 3 release by WEHI-3 cells was also stimulated by retinoic acid in a dose-related fashion. The maximal response was obtained with 10(-8) M-retinoic acid. These results show that retinoic acid, in physiological concentrations, exerts selective effects on interleukin production in vitro, and this stimulation of IL 1 and IL 3 release may explain some of the immunostimulatory effects of retinoids in vivo. Moreover, since IL 1 is known to influence connective tissues and bone, an increase in IL 1 might also explain some of the changes observed in these tissues in vitamin A poisoning and with high-dose retinoid therapy.     

Kinetic analysis of megakaryocyte numbers and ploidy levels in developing colonies from mouse bone marrow cells

Abstract. The kinetics of megakaryocyte formation from mouse bone marrow cells in semi-solid medium was studied directly in the culture dish by staining the cells for acetylcholinesterase after drying the cultures. A WEHI-3 cell-conditioned medium (WEHI-3 CM) was used as a general source of stimulus for megakaryocyte colony formation. The addition of peritoneal exudate supernatant as well as WEHI-3 CM increased the frequency of megakaryocyte colonies detected. Colonies containing acetylcholinesterase-positive cells were first detected at day 3. Maximum numbers of 25–40 megakaryocyte colonies per 10⁵ nucleaet mouse bone marrow cells were observed from days 7 to 11. The mean number of cells within each colony increased progressively with time of culture, and a modal range of 11–20 cells was obtained by day 7. Between 3 and 200 cells per colony were generally detected. A continuous distribution of the number of megakaryocytes per colony suggests that the clonable precursor cells are not synchronized either with respect to maturation stage or with respect to their capability to undergo nuclear endoreduplication. The addition of peritoneal exudate supernatant to the cell cultures increased the DNA levels of megakaryocytes grown in the presence of WEHI-3 CM but did not affect the number of cells per colony. The DNA content of colony megakaryocytes was measured after staining the cells with Feulgen reagent. A modal DNA value of 8 N was observed between days 4 and 7 for megakaryocytes stimulated with WEHI-3 CM. In the presence of both WEHI-3 CM and peritoneal exudate supernatant, the DNA content of megakaryocytes increased with the time of cell culture. Modal DNA values increased from 8 N at days 4 and 5, to 16 N by day 6. In these optimally stimulated cultures, 44% of colony megakaryocytes were 32 N or greater, a proportion higher than in normal bone marrow, but similar to that seen in the marrow of acutely thrombocytopenic animals. It is concluded that megakaryocytopoiesis in cell cultures is not a strictly controlled process with respect to cell division and endomitosis and that when certain culture conditions are employed, megakaryocyte development in vitro might reflect that seen in a stressed animal condition.     

MC3T3-G2/PA6 preadipocytes support in vitro proliferation of hemopoietic stem cells through a mechanism different from that of interleukin 3

Both MC3T3-G2/PA6 preadipocytes and interleukin 3 (IL 3) can support in vitro proliferation of mouse hemopoietic stem cells (CFU-S). We examined whether MC3T3-G2/PA6 cells produce IL 3 and whether a common mechanism might underlie the action of both of these agents. We used cultured mast cells, DA-1 cells, and FDC-P2 cells as the targets of IL 3 and conditioned medium (CM) of WEHI-3 cells as a source of IL 3. MC3T3-G2/PA6 CM did not support the growth of the above cells. IL 3 mRNA was not detected in the preadipocytes. Since CM obtained from the cocultures of bone marrow cells and MC3T3-G2/PA6 cells did not have a significant effect on the growth of the IL 3-dependent cells, none of the bone marrow cells seem to produce IL 3 under the influence of the preadipocytes. When the factor-dependent cells were cocultured with MC3T3-G2/PA6 cells, the former did not survive, whereas mast cells and DA-1 cells intimately associated with the preadipocytes. Even when bone marrow cells, mast cells, and MC3T3-G2/PA6 cells were cocultured, the number of CFU-S increased, but not that of mast cells. These results seem to exclude the possibility of the action of IL 3 in the microenvironment provided by MC3T3-G2/PA6 preadipocytes.     

Evidence for the involvement of the RNA-dependent protein kinase (PKR) in the induction of human cytotoxic T lymphocytes against a synthetic peptide of HIV-1 by regulatory RNA

The studies on the inhibitory effect exerted by Cholera Toxin (CT) on cell growth and proliferation indicate a remarkable heterogeneity of cell response suggesting that the inhibition represents the final event of many different ways or mechanisms . After CT binding, cAMP accumulation may not occur (as in L1210 leukemia cells) or, when occurring (as in SR-4987 stromal cells), may not be coupled with the antiproliferative effect of CT. In WEHI-3B cells CT binds a Gal-GalNac-GM1b receptor and the anticlonogenic effect of CT seems correlated with cAMP accumulation.To demonstrate the central role of cAMP in WEHI-3B cells, starting from the sensitive cell strain we selected and established a clone of WEHI-3B resistant to CT. This revertant clone (WEHI-3B/CT/REV) is currently cultured in the absence of CT and in the proliferation assay shows a dramatic resistance (>46,000 than the parental cells). Stimulation of WEHI-3B/CT/REV cells by cholera toxin failed to enhance cAMP and the ganglioside-CT binding studied on Thin Layer Chromatography (TLC) blots showed that the resistant cells lost the spot correspondent to the migration of Gal-GalNac-GM1b ganglioside. Both the lines respond at the same level to the adenylate cyclase stimulation by forskolin and the incorporation of GM1a did not decrease the resistance of WEHI-3B/CT/REV. These data confirm that Gal-GalNac-GM1b is the most important functional receptor for CT in WEHI-3B cells able to transduce the signal by enhancing cAMP which in turn inhibits cell proliferation (probably by cAMP dependent protein kinase activation). Our study describes the first cell line resistant to CT originated from a susceptible parental strain and provides a new interesting cell model for studying the cAMP dependent mechanisms involved in cell growth regulation.     

IL-1 inhibits B cell differentiation in long term bone marrow cultures

There is evidence that stromal cells are responsive to changes in their external milieu and that this can affect their function. IL-1 has been identified as one mediator that can affect stromal cells by increasing their secretion of CSF. The monokine has also been reported to be a B cell differentiation factor. The purpose of this study was to test the effects of IL-1 on the pattern of hemopoietic cell differentiation by adding IL-1 alpha to myeloid long term bone marrow cultures (MBMC) at the time of their transfer to lymphoid bone marrow culture conditions. This usually results in the cessation of myelopoiesis and the induction of B lymphopoiesis. The addition of 50 U/ml of rIL-1 alpha, but not 10 U/ml, to MBMC at the time of their transfer to lymphoid conditions resulted in a complete inhibition of B cell differentiation and sustained myelopoiesis. To determine whether adherent layer cells contributed to this effect, conditioned medium (CM) was collected from adherent layers treated previously with the antibiotic mycophenolic acid. This depletes the hemopoietic cells from the cultures and retains a purified population of stromal cells. CM from mycophenolic acid- treated adherent layers exposed for 24 h to 50 U/ml of IL-1 was added at volume concentrations of 5, 10, and 25% to MBMC at the time of transfer to lymphoid bone marrow culture conditions and at each feeding thereafter. Expression of the B lineage associated 14.8 Ag and IgM was inhibited on a dose dependent basis, and myelopoiesis was sustained in cultures to which 25% CM had been added. Induction of B lymphopoiesis occurred in cultures to which adherent cell CM not exposed to IL-1 had been added. The CM from the IL-1-treated adherent cells contained CSF, because it promoted the growth of myeloid colonies from fresh marrow or MBMC cells and stimulated the granulocyte-macrophage-CSF sensitive FDC-P1 cell line to proliferate. IL-3 was not present in the CM, because stimulation of the IL-3 sensitive 32D cell line was not observed. The CM from the IL-1-treated adherent cells stimulated thymocytes to proliferate in the presence of PHA. This raised the possibility that the induced CSF may have required IL-1 to mediate their effects in the cultures. However, B lymphopoiesis was inhibited and myelopoiesis maintained upon addition of recombinant granulocyte-, macrophage-, and granulocyte-macrophage-CSF to cultures, indicating that IL-1 or other non-CSF molecules induced by it need not be present.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulation of hexose uptake by haemopoietic cell growth factor occurs in WEHI-3B myelomonocytic leukaemia cells: a possible mechanism for loss of growth control

WEHI-3B myelomonocytic leukaemia cells secrete a haemopoietic cell growth factor (HCGF) which facilitates the proliferation and development of multipotential stem cells and committed progenitor cells. Several cloned, nonleukaemic cell lines (FDC-P cells) are absolutely dependent on HCGF and die in the absence of it. In these cell lines, factor dependence is associated with the ability of HCGF to increase glucose uptake, thereby controlling glycolytic flux and intracellular ATP levels. We have now investigated the effects of HCGF on glucose uptake in WEHI-3B cells. At 20 degrees C 2-deoxyglucose uptake could be stimulated by the addition of HCGF to the extracellular medium. L-glucose uptake was markedly lower than 2-deoxyglucose uptake and did not respond to the addition of HCGF. At 37 degrees C no HCGF stimulation of 2-deoxyglucose uptake was found. However, at this temperature HCGF release from WEHI-3B cells was markedly higher than at 20 degrees C. Our experiments indicate that HCGF stimulates the glucose transport system in both WEHI-3 cells and FDC-P cells. The similarities between the WEHI-3B cell and FDC-P2 cell polypeptide phenotype were investigated using two-dimensional isoelectric focussing/poly-acrylamide gel electrophoresis. This revealed a high degree of correlation between the two cell types in their protein constituents, indicating a close relationship between the normal and leukaemic cells. These similarities between WEHI-3B cells and FDC-P2 cells are considered and their relevance to haemopoiesis and leukaemogenesis is discussed.     

Production of interleukin 1 by adult T cell leukemia (ATL) cell lines

The accessory function for T cell activation and the production of interleukin 1 (IL 1) of adult T cell leukemia (ATL) cell lines were studied in vitro. ATL cell lines such as Hut-102, MT-1, and MT-2 functioned as accessory cells for the stimulation of human T cell proliferative response induced with concanavalin A (Con A) and induced allogeneic mixed lymphocyte reaction. Cell lysates of three ATL cell lines and the culture supernatant of MT-2 cells had activities to stimulate murine thymocyte proliferative response. Then we studied physicochemical properties of the factors produced by MT-2 cells. The m.w. of the factors were approximately 15,000 by Sephacryl S-200 column chromatography, and their isoelectric point values were 5.4 and 4.8 by chromatofocussing technique. No fraction contained interleukin 2 (IL 2) activities to stimulate IL 2-dependent murine cytotoxic T cell line. The thymocyte-stimulating activities of the factors were absorbed with rabbit anti-IL 1 alpha antiserum, but not with anti-IL 1 beta antiserum. Furthermore, messenger RNA extracted from MT-2 cells hybridized to complementary DNA of IL 1 alpha, but not of IL 1 beta, by Northern blot hybridization analysis. The factors from MT-2 cells could stimulate the production of IL 2 and the expression of IL 2 receptors of human T cells in the presence of Con A as well as recombinant IL 1 alpha and IL 1 beta did, and these activities were also blocked by rabbit anti-IL 1 alpha antiserum, but not by anti-IL 1 beta antiserum. These results suggest that the factors produced by MT-2 cells correspond to IL 1 alpha. However, the accessory function of MT-2 cells for T cell activation was not blocked by rabbit anti-IL 1 antiserum. These results suggest that ATL cell lines produce IL 1-like factors, but the accessory function of ATL cell lines for T cell activation is mediated by some other mechanisms rather than by secreted IL 1-like factors.