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S C Clark

Publications and source records attributed to S C Clark.

At least 91 records · Page 5Linked to original sources
PubMed1991

Interleukin-6 production by the blast cells of acute myeloblastic leukemia: regulation by endogenous interleukin-1 and biological implications.

Coordinate production of interleukin-1 beta (IL-1 beta) and granulocyte macrophage-colony stimulating factor (GM-CSF) or IL-6 by the blast cells of acute myeloblastic leukemia (AML) and normal peripheral blood leukocytes have been previously reported (van der Shoot et al.: Blood 74:2081-2087, 1989; Bradbury et al.: Leukemia 4:44-47 1990a, British Journal of Haematology 16:(in press), 1990b; Rodriguez-Cimadevilla et al.: Blood 76:1481-1489, 1990; Schindler et al.: Blood 75:40-47, 1990). In the present study, we show that IL-6 production by AML blasts is up-regulated by endogenously produced IL-1 beta. Neutralization of the endogenous source of IL-1 results in a significant decrease in IL-6 production, as determined by ELISA. Conversely, exposure of AML blasts to IL-1 alpha results in a significant increase in IL-6 production in 10 of 16 patient samples. Antibodies against IL-1 alpha and -beta also cause a drastic decrease in IL-6 and GM-CSF gene expression by the cells, suggesting that cytokine gene expression in AML blasts is driven, at least in part, by endogenous IL-1. The biologic significance of IL-6 production in culture of AML blasts has been addressed using a neutralizing antibody against IL-6. Our data indicate that IL-6 is important for the survival of clonogenic blasts in culture. In contrast, the survival of the total population of blasts is IL-6-independent, as assessed by the integrity of cellular DNA, even in the presence of anti-IL-6. These observations are consistent with the view that AML blasts might be organized as a lineage, with comparable hierarchy as in normal hemopoiesis and, perhaps, increased heterogeneity despite a homogenous appearance (McCulloch and Till: Blood Cells 7:63-77, 1981; Buick and McCulloch: Control of Animal Cell Proliferation. Academic Press, New York, vol. 1, pp. 25-57, 1985). Buick and McCulloch have identified a subpopulation of AML clonogenic cells with stem-cell-like properties, and suggested that the majority of blasts may have undergone a determination-like step. Our data indicate a marked difference in IL-6 requirement for cell survival between precursors and the majority of blasts, suggesting that IL-6 responsiveness may decrease following a determination-like event, i.e., the reduction in proliferative capacity.

Antibodies↗
PubMed1991

Differentiation induction of blast cells in two cases of childhood acute megakaryoblastic leukemia in vitro by interleukin-3 and interleukin-6: an ultrastructural cytochemical study.

Although hematopoietic growth factors influence renewal and differentiation of blast progenitors in acute myelogenous leukemia (AML), morphological maturation of leukemic blasts is thought a rare event, even when cultured in the presence of appropriate growth stimulants. However, light microscopic observation may not be sufficient to clarify precisely the effects of hematopoietic growth factors on the morphological differentiation of leukemic blasts. In this study, using cell culture techniques and electron microscopic cytochemistry for platelet peroxidase (PPO), we studied the effects of interleukin-3 (IL-3) and interleukin-6 (IL-6), both of which are considered to play an important role in normal megakaryocytopoiesis, on the growth and differentiation of blast cells from two patients with childhood acute megakaryoblastic leukemia (AMKL). In both of the two cases, IL-3 stimulated leukemic colony formation in methylcellulose culture, whereas IL-6 showed little such activity. However, in suspension culture, IL-6 was active in promoting megakaryocytic differentiation, although incomplete, as detected by increase in the number of PPO-positive cells, some having demarcation membrane-like structure. This effect was evident in culture with IL-6 alone in one patient, but it was detectable only when IL-6 was used in combination with IL-3 in the other patient. In contrast, IL-3 alone stimulated differentiation towards myeloid but not megakaryocytic lineage. These results indicate that IL-3 and IL-6 have a distinct role in leukemic megakaryocytopoiesis (IL-3 stimulates growth, whereas IL-6 promotes morphological differentiation) and that cooperation between these two cytokines functions most effectively for megakaryocytic differentiation of AMKL cells in a fashion similar to that for normal megakaryocytopoiesis.

Blast Crisis↗
PubMed1991

The role of interleukin-6 in mitogenic T-cell activation: detection of interleukin-2 heteronuclear RNA by polymerase chain reaction.

It has been documented that interleukin-6 (IL-6) supports the proliferation of purified, anti-CD3-stimulated murine T cells. We found that stimulation of human peripheral blood mononuclear cells (PBMCs) with anti-CD3 induced a significant accumulation of IL-6 mRNA, indicating that antigen-mediated T-cell activation may involve IL-6 release from accessory cells. Phytohemagglutinin (PHA) had little effect upon IL-6 gene expression. In keeping with these findings, anti-IL-6 reduced but did not abolish anti-CD3-mediated proliferation of PBMCs, but had no significant effect upon PHA-stimulated proliferation. The addition of recombinant (r) IL-6 enhanced the proliferation of anti-CD3-stimulated PBMCs and increased the accumulation of IL-2 mRNA in PHA-stimulated PBMCs during the first 5 hr of culture. Nuclear run-off experiments did not reveal significant changes in IL-2 transcription in PHA plus rIL-6-treated PBMCs attempting to assume that IL-6 mediates stabilization of IL-2 mRNA. However, monitoring of partially spliced IL-2 mRNA by polymerase chain reaction revealed a clear increase in IL-2 heteronuclear RNA. Thus IL-6 increases the rate of IL-2 transcription which was not detectable by conventional in vitro transcription assays. We conclude that anti-CD3 triggers T-cell proliferation through a process that is partially but not entirely dependent upon release of IL-6. IL-6, in turn, supports IL-2 transcription. Insofar as anti-CD3 mimics antigen-triggered activation of the T-cell receptor complex, IL-6 appears to support the early immune response by augmenting antigen-triggered IL-2 gene expression.

Antibodies, Monoclonal↗
PubMed1991

Interleukin-4 as a growth regulator of clonogenic cells in acute myelogenous leukemia in suspension culture.

Using two complementary culture systems, suspension and clonal cultures, and with a method of graphic display (star diagram), we studied the effects of recombinant human interleukin-4 (IL-4) on leukemic stem cell renewal and differentiation in acute myelogenous leukemia (AML). The interactions between IL-4 and other recombinant human cytokines, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), macrophage CSF (M-CSF) and interleukins-1 alpha, -2, -3, -5, and -6 were also studied. IL-4 alone had significant effects on both self-renewal and differentiation of blast progenitors in some cases; in clonogenic assay, IL-4 stimulated blast colony formation and in one case IL-4 was the most powerful stimulator among the nine growth factors tested. Star diagrams, constructed using the data from both suspension and clonal cultures, showed that IL-4 could influence the balance between self-renewal and differentiation of clonogenic cells. Negative and positive interactions were detected between IL-4 and other cytokines in suspension culture. These results indicate that IL-4 is a cytokine with a potential role in regulating the growth of myeloid leukemic stem cells, and that IL-4 may be useful in treating selected AML patients.

Adult↗
PubMed1991

Effect of rhCSF-1 on human hemopoiesis in long-term cultures from patients with acute myelogenous leukemia.

Bone marrow and/or peripheral blood cells from seven patients with acute myelogenous leukemia (AML) were maintained for 7 weeks in Dexter-type long-term culture (LTC) in order to study the effect of exogenous recombinant human colony-stimulating factor-1 (rhCSF-1) and to quantitate endogenous levels of CSF-1. rhCSF-1 was added every 2 days during the first 3 weeks of culture at 15 ng/ml. In all but one culture, adding rhCSF-1 inhibited putative leukemic hemopoiesis [i.e. decreased numbers of abnormal (blast) colony-forming cells and blasts] and stimulated putative normal hemopoiesis (increased numbers of CFU-GM and macrophages). Our data, however, do not distinguish direct effects of rhCSF-1 on normal or leukemic cells from indirect effects mediated by accessory cells. In cultures with a poorly formed adherent layer (all derived from patients classified as M5), the endogenous levels of CSF-1 were lower than those in cultures with a good (confluent) adherent layer, indicating that the levels of CSF-1 in LTC from AML patients positively correlate with the formation of the adherent layer. Our data indicate that CSF-1 is an important modulator of human hemopoiesis in LTC established from AML bone marrow or peripheral blood, and that rhCSF-1 might be valuable for purging leukemic cells in LTC established from AML patients' bone marrow or peripheral blood for autologous transplantation.

Hematopoiesis↗
PubMed1991

Establishment of a novel heterotransplantable acute lymphoblastic leukemia cell line with a t(17;19) chromosomal translocation the growth of which is inhibited by interleukin-3.

A cell line, designated HAL-01, was established from the blood cells of a patient with acute lymphoblastic leukemia (ALL) with a myeloid-associated marker. Both the cell line and the patient's fresh leukemia cells had the chromosomal translocation t(17;19)(q21;p13). Morphologically and cytochemically, the cells were lymphoid in appearance. Immunophenotyping of the donor's leukemia cells revealed that they express B lineage antigens (CD10+, CD19+, CD20+, CD22+); the myeloid-associated antigen (CD13) detected in the donor's leukemia cells was not expressed by the established cell line. The HAL-01 cells have a rearrangement of the immunoglobulin heavy chain gene, while the T-cell receptor beta-chain genes remain in the germline configuration. The gene encoding the binding proteins for the kappa-light chain enhancer (kappa E2), which is involved in pre-B-ALL cells with the t(1;19) (q23;p13) translocation, is not rearranged in the cell line. The HAL-01 cells were transplantable into the peritoneum of untreated nude mice where they grew as an ascites tumor. The growing tumor cells also infiltrated lymph nodes, liver, spleen, kidney, and bone marrow without exhibiting a particular change in the morphology of the neoplastic cells. Clonogenic assay in methylcellulose culture demonstrated that the proliferation of the HAL-01 cells was suppressed by interleukin-3 (IL-3) in a dose-dependent fashion, with maximum inhibition occurring at concentrations greater than 100 U/ml. Treatment with IL-3 reduced the number of viable cells as well as induced morphological changes without concomitant changes in cytochemical reactions or immunophenotypic expression. Reduction of 3H-thymidine incorporation by exposure of IL-3 was blocked by the pretreatment of neutralizing anti-IL-3 antibody, but not by neutralizing anti-TGF-beta antibody. Thus, HAL-01 is a unique ALL cell line exhibiting proliferative suppression by IL-3 that may prove useful in studying the interactions of cytokines in ALL.

Adolescent↗
PubMed1990

Growth factor-dependent differentiation along the myeloid and lymphoid lineages in an immature acute T lymphocytic leukemia.

Bone marrow cells from a child with an immature (CD2+, CD5+, CD7+) acute T lymphocytic leukemia (T-ALL) were cultured in the presence and absence of human rIL-2, IL-3, or granulocyte-macrophage (GM)-CSF. Cells cultured without growth factors failed to divide and those initiated in the presence of IL-2 or GM-CSF underwent maturation and terminal T lymphoid or myelomonocytic differentiation, respectively. In contrast, a permanent growth factor-dependent cell line, designated TALL-103/3, was established upon culture in IL-3. The TALL-103/3 cells gradually lost the T cell-specific markers and acquired a myeloid phenotype (CD15+, CD33+). Switching of the IL-3-dependent cells at an early passage to medium containing only human rIL-2 resulted in the establishment of a subline, named TALL-103/2, with a T lymphoid phenotype (CD3+, CD8+, TCR-gamma delta +, CD7+). The TALL-103/2 cells strictly require IL-2 for growth, are irreversibly committed to the lymphoid lineage, and cannot survive in the presence of any other hemopoietic growth factor tested so far. In contrast, the IL-3-dependent TALL-103/3 cells could be adapted to grow in synthetic (serum-free) medium also in the presence of either GM-CSF or IL-5, in which they retain a myeloid phenotype. Interestingly, after 18 mo in culture in IL-3, the TALL-103/3 cells can still be phenotypically converted to the lymphoid lineage upon addition of IL-2, thus maintaining its bipotentiality. Despite the marked phenotypic differences, the TALL-103/2 and TALL-103/3 cell lines show the same karyotypes with multiple abnormalities present in the primary malignant clone and have identical rearrangements of the TCR-gamma and -delta loci, thus confirming their derivation from a common precursor cell. Together, these findings indicate that the phenotype of immature T-ALL cells can be drastically modified by the presence of specific hemopoietic growth factors in the environment, leading to either lymphoid or myeloid lineage commitment while leaving their karyotype and genotype intact.

Antigens, CD↗
PubMed1990

Human thymic epithelial cells produce IL-6, granulocyte-monocyte-CSF, and leukemia inhibitory factor.

The development of conditions for culturing normal human thymic epithelial (TE) cells free from contaminating stromal cells has allowed us to characterize a number of cytokines produced by TE cells. Using cDNA probes for human IL-6, granulocyte-monocyte-CSF, and leukemia inhibitory factor (LIF), we identified mRNA for these cytokines by RNA blot analysis of total RNA preparations derived from TE cells. We demonstrated that TE cells produced IL-6 transcripts and that TE cell culture supernatants contained IL-6 biologic activity, as determined by the ability to support proliferation of the T1165 plasmacytoma line. The 1.0-kilobase (kb) transcript of granulocyte-monocyte-CSF was also detected in TE cell-derived total RNA. TE cell culture supernatants contained LIF activity, as determined by proliferation of the murine cell line DA-1a, and a 4.0-kb LIF transcript was detected in TE cell-derived total RNA preparations. The 4.0-kb LIF transcript from TE cell-derived total RNA corresponded in size to the LIF transcripts in PMA-activated T lymphocytes. Thus, using biologic assays and RNA blot analysis, we demonstrated that cultured normal human TE cells produced both immunoregulatory cytokines and cytokines that drive various differentiation stages of human hematopoiesis. Our findings support the hypothesis that TE cells may play a role in providing cytokines that are important for the proliferation and differentiation of hematopoietic precursor cells that migrate to the thymus during fetal and postnatal human thymic development.

Antigens, CD7↗
PubMed1990

Comparison of the effects of IL-3, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor in supporting monocyte differentiation in culture. Analysis of macrophage antibody-dependent cellular cytotoxicity.

Cultured human monocytes undergo a process of differentiation and maturation lasting 5 to 10 days that ultimately leads to the appearance of large macrophage-like cells. This differentiation is growth factor dependent: of all the cytokines tested, only macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF (GM-CSF), and IL-3 proved capable of supporting the differentiation and the long term survival of the macrophage-like cells. Although all three cytokines yield cells with macrophage characteristics, cells developed in M-CSF have features distinct from those matured in either IL-3 or GM-CSF. At the morphologic level, the M-CSF-supported monocyte cultures yield elongated, spindle-shaped cells whereas those supported with IL-3 or GM-CSF yielded round cells with distinct nuclei. All three macrophage populations expressed similar levels of HLA-DR, CD11b, and CD11c, but the M-CSF-treated cultures yielded more CD14+ and CD16+ (Fc gamma RIII) cells. All three cell populations developed capacity for antibody-dependent cellular cytotoxicity (ADCC) as well as antibody-independent cytotoxicity with peak activity achieved after 8 to 12 days in culture. ADCC capacity developed earliest and the level of activity was usually greatest in the M-CSF-treated cultures, possibly correlating with the higher level of expression of CD16. Our findings indicate that any of these cytokines, but particularly M-CSF, may be useful clinically in enhancing the tumoricidal capacity of tumor-specific mAb through augmentation of macrophage capacity for ADCC.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1990

Human P40 T-cell growth factor (interleukin-9) supports erythroid colony formation.

Because human P40 T-cell growth factor, tentatively designated interleukin-9 (IL-9), was isolated through its ability to stimulate a human IL-3-dependent leukemic cell line (M-O7E), we tested the ability of IL-9 to support the growth and differentiation of normal hematopoietic progenitor cells from peripheral blood and bone marrow. Although the M-O7E cell line was derived from a patient with megakaryoblastic leukemia, IL-9 has not proved to be a growth or maturation factor for megakaryocytes, but instead has proved to be effective in supporting the development of erythroid bursts (BFU-E) in cultures supplemented with erythropoietin. Using highly purified progenitors from peripheral blood, IL-3 showed a BFU-E plating efficiency of 46% compared with 20% for IL-9. Because of the purity of these cell preparations and the low cell density in culture, IL-9 is likely to interact directly with erythroid progenitors. Analysis of mixing experiments and of the morphology of the BFU-E in culture indicated that IL-9 interacts preferentially with a relatively early population of IL-3-responsive BFU-E. In cultures of human bone marrow or cord blood, IL-9 selectively supported erythroid colony formation, while IL-3 and granulocyte/macrophage colony-stimulating factor additionally yielded granulocyte/macrophage colonies. Therefore, IL-9 represents a new T cell-derived cytokine with the potential for selectively stimulating erythroid development in the hematopoietic system.

Antigens, CD↗
PubMed1990

Synergistic and antagonistic effects of IL-1 alpha and IL-4, respectively, on the IL-2-dependent growth of a T cell receptor-gamma delta+ human T leukemia cell line.

The TALL-103/2 cell line was derived from an immature acute T lymphocytic leukemia with T-myeloid differentiating capacity. The leukemic cells were first expanded in recombinant human IL-3 in which they acquired a myeloid phenotype, and subsequently were adapted to grow in human rIL-2 in which they became lymphoid committed. The TALL-103/2 cell line expresses only T cell-specific differentiation Ag (CD2, CD3, CD7, and CD8) but has retained the CD33 myeloid Ag originally present on the IL-3 expanded population. By using mAb directed at the TCR-alpha beta or specific for framework determinants on human TCR-gamma and -delta chains, the TALL-103/2 cells were shown to be WT31-, TCR delta 1+, TCS-1+, and Ti gamma A-, thus representing a T cell subset expressing the nondisulfide-linked form of the TCR-gamma delta. The TALL-103/2 cells have been maintained for more than 1 y in the presence of human rIL-2 on which they are strictly dependent. Chemical cross-linking and immunofluorescence studies indicate the presence of both high and intermediate affinity IL-2R on the TALL-103/2 cells. Whereas mAb antiTac and H-31 with reactivity to the IL-2R alpha-chain (p55) compete only partially for the IL-2-induced proliferation of these cells, mAb TU27, specific to the IL-2R beta-subunit (p75), inhibits such growth completely even at high concentrations of IL-2. The interactions of the two T cell-stimulating factors IL-1 and IL-4 on the IL-2-dependent growth of TALL-103/2 cells were investigated. IL-1 alpha synergizes with IL-2 in supporting the short and long term growth of this cell line, whereas IL-4 abrogates its growth. These effects are, at least in part, due to the modulation of IL-2R expression induced by the two lymphokines. Functionally, the TALL-103/2 cells display MHC-nonrestricted cytotoxic activity that is significantly enhanced by addition of either IL-4, IL-6, or IFN-gamma. Because of its properties and its stable requirement for IL-2 for continuous growth, this T lymphocytic leukemia-derived cell line represents an interesting model to analyze ontogeny and function of leukemic T cells.

Antigens, CD↗
PubMed1990

Transcriptional modulation of human IL-6 gene expression by verapamil.

Calcium channel-blocking agents interfere with the initial increase of cytosolic calcium that follows mitogenic stimulation of T lymphocytes. In cultures of mitogen-stimulated PBMC, verapamil also blocked T cell accumulation of cytoplasmic IL-2-encoding mRNA. In sharp contrast, the addition of verapamil to PHA and PMA-stimulated PBMC augmented the mitogen-stimulated increases in nuclear transcription of IL-6-encoding mRNA, steady state levels of IL-6 encoding mRNA, and release of IL-6 bioactivity. These experiments indicated that an increased IL-6 transcriptional rate rather than stabilization of transcripts accounted for the increased cytoplasmic IL-6 mRNA levels and subsequent expression of IL-6 bioactivity. These effects were not produced by nicardipine, another potent calcium channel blocker, or EGTA. We suggest that a non-calcium-dependent, IL-6 regulatory factor, absent or inactive in verapamil-treated cultures, inhibits IL-6 gene activation in mitogen-stimulated PBMC. Failure to express this inhibitory factor would result in IL-6 gene superinduction at a transcriptional level.

Blotting, Northern↗
PubMed1990

Leukemia inhibitory factor differentiation-inhibiting activity/human interleukin for DA cells augments proliferation of human hematopoietic stem cells.

Leukemia inhibitory factor (LIF)/differentiation-inhibiting activity (DIA)/human interleukin for DA cells (HILDA) is a cytokine with biologic activities involving a variety of different types of target cells. Here we have tested LIF/DIA for possible effects on the growth and differentiation of normal human hematopoietic cells in culture. As a single agent, LIF/DIA had no effect on colony formation by CD34-positive human bone marrow cells. However, LIF/DIA was as effective as either interleukin-6 (IL-6) or granulocyte colony-stimulating factor (G-CSF) in the enhancement of IL-3-dependent colony formation of very primitive blast colony-forming cells. Studies using neutralizing antibodies against IL-6 or G-CSF demonstrated that this was not due to induction in culture of either of the other known synergistic factors for blast cell colony formation. A 1-day delay in the time course of appearance of blast cell colonies grown in the presence of LIF/DIA relative to those grown in the presence of IL-6 suggests that the different synergistic factors may operate through different mechanisms, although we cannot rule out that high doses of LIF/DIA might yield accelerated blast cell colony formation. Our findings provide evidence that LIF/DIA may play an important role, along with IL-6 and G-CSF, in the regulation of early hematopoietic stem cells.

Antibodies↗
PubMed1990

Serum-free culture of enriched hematopoietic progenitors reflects physiologic levels of fetal hemoglobin biosynthesis.

Adult erythroid progenitors produce significantly higher fetal hemoglobin (HbF) levels in cultures containing fetal calf serum (FCS) and erythropoietin (Ep) than in vivo. The precise mechanisms for this increased HbF production in culture have not been elucidated. We examined HbF biosynthesis by enriched human progenitors in serum-free (SF) culture. We measured globin chain biosynthesis by combination of isoelectric focusing and autoradiography and examined percent nucleated erythrocytes containing HbF (%FNRBC) using microscopic immunodiffusion. CD34 (My10)-positive marrow cells from a normal subject yielded an almost negligible amount of gamma-globin in SF culture stimulated by 100 U/mL interleukin-3 (IL-3) and 2 U/mL Ep, while corresponding FCS culture revealed significant gamma-globin biosynthesis. The %FNRBC of the erythroid bursts in SF cultures derived from nine normal adults (2.0% +/- 0.9% F cells) was 3.0% +/- 3.4%, while in FCS culture, it was 25% +/- 12% (mean +/- SD). Dosages of IL-3 between 10 and 10,000 U/mL did not increase %FNRBC in FCS of SF conditions. Mean Hb contents of nucleated erythrocytes (NRBC) assayed by microdensitometry of pericellular immunoprecipitate were similar in FCS and SF cultures. The number of erythroid bursts per 2 x 10(3) CD34-positive marrow cells was 48 +/- 20 in FCS and 36 +/- 12 in SF cultures. In two experiments, progenitors grown for 7 days under SF conditions were isolated and recultured in either SF or FCS conditions for 7 days, and the resulting erythroid bursts were analyzed for FNRBC. The bursts that had been returned to FCS cultures yielded values of %FNRBC intermediate between those obtained from progenitors grown entirely in SF or FCS cultures, indicating that serum effect is not solely due to growth selection for certain subpopulations of erythroid burst-forming units. This experiment also demonstrated that the factors present in serum responsible for HbF augmentation act at both early and late stages during erythroid burst development. SF culture of peripheral blood progenitors of one subject with heterocellular hereditary persistence of fetal hemoglobin (HPFH) yielded elevated levels of FNRBC (19% +/- 5%) that accurately reflected the F cell (18%) of the circulating blood. Similarly, FNRBC in cultures of progenitors from one umbilical cord blood sample (86% F reticulocytes) was 87 +/- 3% FNRBC. The SF culture for enriched human progenitors, which nearly reflects the physiologic HbF programs of the donor, should facilitate studies of the exact mechanisms of postnatal reactivation of HbF production.

Bone Marrow↗
PubMed1990

Characterization of granulocyte-macrophage colony-stimulating factor receptor on the blast cells of acute myeloblastic leukemia.

Iodinated granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to document the specific binding of GM-CSF to all acute myeloblastic leukemia (AML) samples examined in the present study. There was some heterogeneity in the number of GM-CSF binding sites per cell. To determine whether the low level of binding to some patient samples may be attributed to receptor occupancy by an endogenous source of GM-CSF, we devised an acid wash procedure that could remove surface-bound GM-CSF without affecting receptor properties. We thus document that GM-CSF specific binding to AML blasts before or after acid wash was the same, indicating that the observed heterogeneity in binding is not the result of receptor occupancy by an endogeneous source of GM-CSF. Saturation analyses are in favor of the presence of two classes of binding sites on AML blasts: a high-affinity receptor that binds GM-CSF with a dissociation constant (kd) of 3 to 73 pmol/L and a second class of low-affinity receptor that binds GM-CSF with a kd of 1 to 10 nmol/L. Binding studies with two established cell lines KG-1, and IRCM-8 also showed the presence of two classes of binding sites with high and low affinities. Analysis of GM-CSF titration curves in culture indicate that the median effective concentration required for stimulation of blast colony formation (EC50 = 5-36 pmol/L) were in the range of the kd of the high-affinity binding site, suggesting that this high-affinity binding site mediates the proliferative response.

Cell Division↗
PubMed1990

Correlations and interactions in the production of interleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF.

Interleukin-6 (IL-6) shares several biologic properties with IL-1, including hematopoietin-1 activity and stimulation of T cells. Because many of their biologic activities overlap, we developed and used a specific radioimmunoassay (RIA) for IL-6 to compare production of this cytokine on a molar basis with that of IL-1 alpha, IL-1 beta, and tumor necrosis factor (TNF)alpha. The RIA correlated well with the hybridoma bioassay for IL-6 (r = .87, P less than .001). Freshly isolated human peripheral blood mononuclear cells (PBMC) cultured in the absence of stimuli did not produce IL-6 in most cases. Kinetics of secretion and cell-association of IL-6 were studied. In contrast to IL-1 alpha but similar to TNF, IL-6 was almost entirely secreted into the extracellular fluid. Incubation with different stimuli (lipopolysaccharide [LPS], phytohemagglutinin [PHA], Staphylococcus epidermidis, or IL-1 alpha) resulted in production of IL-6. However, on a molar basis PBMC produced approximately two to three times less IL-6 than IL-1 alpha, IL-1 beta, or TNF, regardless of the stimulus. The amount of IL-6 produced from PBMC was consistent when measured in the same subjects six time during a 12-week period. In a cohort of 38 donors, the coefficient of variation for IL-6 production was .32, compared with .92 for IL-1 beta and .96 for TNF. Comparing cytokine production by PBMC, there was a significant correlation between IL-6 and IL-1 beta (r = .72) and between IL-6 and TNF (r = .66). IL-6 did not stimulate IL-1 beta or TNF production, but suppressed IL-1 beta and TNF production induced by LPS or PHA by 30% (P less than .01). This suppression of IL-1 beta and TNF by IL-6 appears to be on the level of transcription.

Biological Assay↗