Patients with systemic lupus erythematosus with high plasma levels of sFas risk relapse.

OBJECTIVE: We related soluble Fas (sFas) levels to the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) in a longitudinal series of plasma samples of patients with SLE to evaluate the relation between excessive production of sFas and disease activity. METHODS: We generated 21 monoclonal antibodies against Fas. Two of these were used to develop and validate a sensitive sandwich ELISA for the longitudinal analysis of sFas levels in plasma of 30 patients and 25 controls. RESULTS: At the start of followup, a significant elevation (p<0.0001) was found in sFas levels in SLE (1167+/-347 pg/ml sFas) compared to controls (618+/-98 pg/ml sFas). Also, at the start of the followup a significant difference (p = 0.0028) existed between patients who were going to have a relapse (1236+/-402 pg/ml sFas) during followup and patients who were not (809+/-276 pg/ml sFas). While sFas did not fluctuate with disease activity in individual patients, we found a strong correlation (r = 0.75, p<0.0001) between sFas and SLEDAI, but only at the time of relapse, when we analyzed the patients as a group. CONCLUSION: In individual patients with SLE, sFas does not fluctuate with disease activity. However, patients with high plasma levels of sFas are at risk of relapse.

Soluble Fas/APO-1 in tumor cells: a potential regulator of apoptosis?

Fas/APO-1, a member of the NGF/TNF receptor superfamily expressed on the cell-surface of normal and malignant cells, is known to induce cell death by apoptosis. In the present study, we have investigated Fas/APO-1 gene defects in a human osteosarcoma cell line resistant to the apoptosis-inducing effects of anti-Fas. cDNA cloning and sequencing revealed that these cells contained both 'authentic' and mutant Fas/APO-1 containing a 63 base pair in-frame deletion spanning the transmembrane domain, designated DFas/APO-1. Direct evidence for the existence of a soluble Fas/APO-1 protein was obtained by immunoprecipitation and Western blotting. Taken together with prior studies demonstrating a role for Fas/APO-1 and Fas ligand, respectively, in tumor target cell killing by cytotoxic T-lymphocytes, production of soluble Fas/APO-1 might have significant implications in malignant disease pathogenesis.

Human serum megakaryocyte colony-stimulating activity appears to be distinct from interleukin-3, granulocyte-macrophage colony-stimulating factor, and lymphocyte-conditioned medium.

Sera from patients with bone marrow megakaryocyte aplasia are a rich source of megakaryocyte colony-stimulating activity (Meg-CSA). Other biologic materials exhibiting Meg-CSA include phytohemagglutinin-stimulated human lymphocyte-conditioned medium (PHA-LCM), recombinant interleukin-3 (IL-3), and recombinant granulocyte macrophage colony-stimulating factor (GM-CSF). Neutralizing antisera to both recombinant IL-3 and GM-CSF were used to evaluate the relationship among these sources of Meg-CSA. Varying dilutions of IL-3 and GM-CSF antisera were tested in plasma clot cultures of normal human peripheral blood megakaryocyte progenitors optimally stimulated by either IL-3 (1 U/mL), GM-CSF (1 U/mL), PHA-LCM (2.5% to 5% vol/vol), or aplastic human serum (10% vol/vol). IL-3 antiserum at dilutions up to 1/2,000 totally abrogated megakaryocyte colony growth stimulated by IL-3. A 1/500 dilution of GM-CSF antiserum completely eliminated GM-CSF-induced megakaryocyte colony development. A combination of anti-IL-3 and anti-GM-CSF, each at a 1/500 dilution, inhibited all megakaryocyte colony growth stimulated by optimal concentrations of IL-3 and GM-CSF together. There was no neutralizing crossreactivity between the IL-3 and GM-CSF antisera. At maximally neutralizing concentrations, IL-3 antiserum inhibited 66% of the megakaryocyte colony growth stimulated by PHA-LCM. Residual megakaryocyte colony growth was eliminated by the addition of a 1/500 dilution of anti-GM-CSF.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification through chemical cross-linking of distinct granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors on myeloid leukemic cells, KG-1.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) each bind specifically to a small number of high-affinity receptors present on the surface of the cells of the acute myelogenous leukemia line, KG-1. Through chemical cross-linking of IL-3 and GM-CSF to KG-1 cells, we identified distinct binding proteins for each of these cytokines with approximate molecular masses of 69 and 93 Kd, respectively. Although these two binding proteins are distinct, GM-CSF and IL-3 compete with each other for binding to KG-1 cells. Other cell lines, which express receptors for either factor but not for both do not display this cross-competition for binding with IL-3 and GM-CSF. These findings imply that distinct IL-3 and GM-CSF binding proteins are expressed on the cell surface and that an association exists between these proteins on KG-1 cells.

Proliferative effect of interleukin-3 on normal and leukemic human B cell precursors.

We have examined the influence of recombinant, human IL-3 on normal and leukemic B cell precursors. CD10/CALLA+ leukemic B cell precursors, the pre-B cell line BLIN-1, and surface IgM-B cell precursors isolated from fetal bone marrow all responded to IL-3. This IL-3 response was dose-dependent and could be abrogated by a rabbit anti-IL-3 antiserum. Binding studies using radiolabeled IL-3 revealed the presence of approximately 200 high affinity IL-3 receptors on the BLIN-1 cell line, with a KD of 150pM. We conclude that normal and leukemic human B cell precursors express functional IL-3 receptors, extending the biologic range of this hematopoietin to the lymphoid lineage.

Leukemic B-cell precursors express functional receptors for human interleukin-3.

The purpose of this study was to analyze the expression of functional interleukin-3 (IL-3) receptors on leukemic B-cell precursors (BCPs) from 12 BCP acute lymphoblastic leukemia (ALL) patients and five BCP ALL cell lines. The specific binding of biosynthetically labeled 35S-recombinant (r) IL-3 to freshly obtained leukemic marrow blasts was initially investigated. In five of 12 BCP ALL cases, the binding of 35S-rIL-3 was markedly blocked by excess cold rIL-3, and the percentage of inhibitable binding ranged from 53% to 78% (mean +/- SE = 65% +/- 4%). In these cases, the cell-bound radioactivity ranged from 146 cpm/10(7) cells to 1,433 cpm/10(7) cells (mean +/- SE = 627 +/- 250 cpm/10(7) cells), indicating that 1 to 14 femtomole (mean +/- SE = 6 +/- 2 fms) of [35S]rIL-3/10(7) cells were specifically bound (= 60 to 840 molecules per cell). rIL-3 stimulated the proliferative activity of leukemic BCPs in a dose-dependent fashion without inducing differentiation, and the half-maximal stimulatory activity was observed at a concentration of 17 to 34 pmol/L. Fluorescence-activated cell sorter (FACS)-isolated virtually pure populations of CD10+CD19+ leukemic BCPs from two BCP ALL patients, as well as from two of five BCP ALL cell lines, showed a marked proliferative response to highly purified rIL-3, providing formal evidence that the observed IL-3 responses were not mediated by accessory cells. There was a high correlation between [35S]rIL-3 binding and proliferative response in colony assays, indicating that functional IL-3 receptors were detected in ligand binding assays. Scatchard plot analysis of the specific equilibrium binding data for IL-3-responsive leukemic BCPs from one BCP ALL patient and two BCP ALL cell lines yielded a straight linear regression line, indicating the existence of a single class of 60 to 210 high-affinity IL-3 binding sites/cell. The calculated apparent affinity constant (Ka) values ranged from 3.6 x 10(9) to 5.9 x 10(9) mol/L-1. Hence, the concentration of IL-3 required to produce 50% maximal receptor occupancy (Kd) was in the range of 168 to 280 pmol/L. These concentrations are approximately tenfold higher than those required to induce 50% maximal proliferative response from leukemic BCPs in colony assays, indicating that low receptor occupancy is sufficient for growth stimulation of leukemic BCPs by rIL-3. In comparison, less than 10 to 20 IL-3 molecules/cell were bound to IL-3 unresponsive leukemic BCPs even when the concentration of free-[35S]rIL-3 was as high as 2 nmol/L.(ABSTRACT TRUNCATED AT 400 WORDS)

Recombinant gibbon interleukin-3 stimulates megakaryocyte colony growth in vitro from human peripheral blood progenitor cells.

Gibbon interleukin-3 (rIL-3) has recently been cloned and found to have a high degree of homology with the human IL-3 molecule. In this investigation, we evaluated the effects of gibbon rIL-3 on normal human peripheral blood megakaryocyte progenitor cell growth in vitro. Gibbon rIL-3 exhibited substantial megakaryocyte colony stimulatory activity (Meg-CSA), supporting peak colony numbers at a concentration of 1 U/ml. Megakaryocyte colony growth induced by rIL-3 reached 58% of the maximum achieved with the active, Meg-CSA-containing protein fraction of aplastic canine serum. Increasing gibbon rIL-3 concentrations also stimulated a 4-5-fold increase in megakaryocyte colony size and resulted in a decrease in geometric mean megakaryocyte ploidy. Ploidy values fell from 8.5N +/- 1.4 (+/- SEM) at an rIL-3 concentration of 0.1 U/ml to a minimum of 2.9N +/- 0.3 at 10 U/ml. In the presence of rIL-3 at 1.0 U/ml, megakaryocyte colony growth was linear with cell plating density and the regression line passed approximately through the origin. The effects of rIL-3 on megakaryocyte colony growth were independent of the presence of T-lymphocytes in the cultures. Cross-species evaluation of murine and gibbon IL-3 indicated that its bioactivity is species restricted. Murine IL-3 did not support colony growth from human megakaryocyte progenitors and gibbon rIL-3 showed no activity in stimulating acetylcholinesterase production by murine bone marrow cells. Gibbon rIL-3 is a potent stimulator of the early events of human megakaryocyte progenitor cell development promoting predominantly mitosis and early megakaryocytic differentiation.

Specific binding, internalization, and degradation of human recombinant interleukin-3 by cells of the acute myelogenous, leukemia line, KG-1.

We have studied the interaction of 35S-labeled recombinant IL-3 with the acute myelogenous leukemia cell line, KG-1. 35S-IL-3 bound to these cells in a time dependent, saturable, and specific manner at 4 degrees C. Scatchard transformation of binding isotherms demonstrated the existence of a small number (200) of binding sites, with an apparent dissociation constant of 70-105 pM. After a temperature shift from 4 degrees C to 37 degrees C, surface-bound 35S-IL-3 was rapidly internalized and processed into a trichloroacetic acid soluble form that was released into the medium. Experiments to address the specificity of the IL-3 binding site revealed that neither human IL-2, M-CSF, erythropoietin, transferrin, bovine insulin, nor murine nerve growth factor compete with IL-3 for binding to KG-1 cells. Both human and gibbon recombinant IL-3 and, surprisingly, human recombinant GM-CSF effectively competed the binding of the labeled IL-3 to these cells at 4 degrees C. The competition by GM-CSF was found to be concentration dependent, but much higher concentrations were required to achieve the levels obtained with IL-3. These results suggest that GM-CSF may also interact with the high-affinity IL-3 binding site on KG-1 cells or, alternatively, that GM-CSF binding to its own receptor may decrease the affinity of the IL-3 receptor for its ligand.

Binding and internalization of recombinant human erythropoietin in murine erythroid precursor cells.

Erythropoietin (EPO) biosynthetically labelled with [35S]cysteine was produced from Chinese hamster ovary (CHO) cells containing amplified copies of human EPO cDNA. The glycosylated recombinant [35S]EPO, purified to virtual radiochemical homogeneity, was biologically active. We studied the interaction of this labeled recombinant EPO with erythroid precursor cells from mice made anemic with phenylhydrazine. The [35S]-labeled molecule bound to erythroid precursors in a time- and temperature-dependent manner. The binding was specific for EPO, and neither insulin, transferrin, epidermal growth factor, nor multiplication stimulating activity could compete for EPO binding sites. In the presence of 0.2% sodium azide, which blocks 80% to 90% of internalization, the recombinant molecule bound with an apparent Kd of 750 pmol/L and 100 to 200 binding sites per cell at 37 degrees C. Asialo-EPO was a more effective competitor than sialated EPO for the available binding sites. Thus, the enhanced biological specific activity of asialo-EPO could result from its enhanced binding affinity. We also studied recombinant human EPO labeled with 125I and found that it also bound to the erythroid cells in a saturable and specific manner. After 90 minutes of incubation at 37 degrees C, most of the bound [35S]EPO was internalized, whereas most of the [125I]EPO remained on the cell surface. The reduced internalization of the iodinated molecule could account for the previously reported functional deficit associated with iodination.

Dependence of highly enriched human bone marrow progenitors on hemopoietic growth factors and their response to recombinant erythropoietin.

Human bone marrow cells were sequentially fractionated by three negative selection steps to remove adherent cells and Fc receptor-bearing cells, followed by immune adsorption (panning) to deplete maturing cells that react with a panel of monoclonal antibodies. This nonadherent Fc receptor and antibody negative fraction could be further enriched by a positive selection "panning" step, using an antibody to HLA-DR antigen; 12-27% of the cells formed erythroid burst-forming unit (BFU-E), erythroid colony-forming unit, granulocyte-monocyte colony-forming unit, and erythroid and granulocyte and/or monocyte colony-forming unit-derived colonies with recovery of 0.5-1% of the cells and 20-100% of the colony-forming cells. Sequential fractionation resulted in increasing dependence of a subset of BFU-E-derived colonies on exogenous burst-promoting activity (BPA) for proliferation in culture, but the most enriched progenitor fraction still contained a proportion of accessory cell or BPA-independent BFU-E that responded to either natural or biosynthetic erythropoietin when added to cultures on day 0 in the absence of BPA. If the addition of erythropoietin was delayed until day 3, the data suggest that this population of BFU-E either died or became unresponsive to erythropoietin. Delayed addition of erythropoietin to cultures of enriched progenitors provided a sensitive BPA assay, since BPA-independent but erythropoietin-responsive BFU-E were eliminated. The surviving BFU-E that were dependent for their proliferation on the presence of both BPA and erythropoietin showed a characteristic dose response to increasing BPA concentrations.