Monomeric and dimeric forms of soluble receptors can differ in their neutralization potential.

Recombinant soluble forms of transmembrane receptors can be produced in monomeric and dimeric versions. Binding affinity and neutralization potential of these different forms of soluble receptors depend on the quaternary structure of their ligands. Monomeric ligands will be bound with equal affinity by both forms, whereas trimeric ligands, e.g. members of the tumor necrosis factor family of ligands, interact with much higher affinity with dimeric soluble receptors than with monomeric ones.

Combination protocols of cytokine therapy with interleukin-3 and granulocyte-macrophage colony-stimulating factor in a primate model of radiation-induced marrow aplasia.

Single cytokine therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) has been shown to be effective in decreasing the respective periods of neutropenia and thrombocytopenia following radiation- or drug-induced marrow aplasia. The combined administration of IL-3 and GM-CSF in normal primates suggested that a sequential protocol of IL-3 followed by GM-CSF would be more effective than that of GM-CSF alone in producing neutrophils (PMN). We investigated the therapeutic efficacy of two combination protocols, the sequential and coadministration of recombinant human IL-3 and GM-CSF relative to respective single cytokine therapy, and delayed GM-CSF administration in sublethally irradiated rhesus monkeys. Monkeys irradiated with 450 cGy (mixed fission neutron:gamma radiation) received either IL-3, GM-CSF, human serum albumin (HSA), or IL-3 coadministered with GM-CSF for days 1 through 21 consecutively postexposure, or IL-3 or HSA for days 1 through 7 followed by GM-CSF for days 7 through 21. All cytokines and HSA were injected subcutaneously at a total dose of 25 micrograms/kg/d, divided twice daily. Complete blood counts (CBC) and platelet (PLT) counts were monitored over 60 days postirradiation. The respiratory burst activity of the PMN was assessed flow cytometrically, by measuring hydrogen peroxide (H2O2) production. Coadministration of IL-3 and GM-CSF reduced the average 16-day period of neutropenia and antibiotic support in the control animals to 6 days (P = .006). Similarly, the average 10-day period of severe thrombocytopenia, which necessitated PLT transfusion in the control animals, was reduced to 3 days when IL-3 and GM-CSF were coadministered (P = .004). The sequential administration of IL-3 followed by GM-CSF had no greater effect on PMN production than GM-CSF alone and was less effective than IL-3 alone in reducing thrombocytopenia. PMN function was enhanced in all cytokine-treated animals.

A GM-CSF/IL-3 fusion protein promotes neutrophil and platelet recovery in sublethally irradiated rhesus monkeys.

The effects of a human GM-CSF/IL-3 fusion protein (PIXY321) were examined in a primate model of rebound hematopoiesis following sublethal irradiation. The results demonstrated an enhanced rate of both neutrophil and platelet regeneration, as well as functional activation of circulating neutrophils.

Mast cell growth factor (C-kit ligand) in combination with granulocyte-macrophage colony-stimulating factor and interleukin-3: in vivo hemopoietic effects in irradiated mice compared to in vitro effects.

In the presence of hemopoietic cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), mast cell growth factor (MGF; also known as steel factor, stem cell factor, and c-kit ligand) has proven to be a potent hemopoietic regulator in vitro. In these studies, we examined the in vivo effects of MGF in combination with GM-CSF or GM-CSF plus IL-3. Effects were based on the ability of these cytokines to stimulate recovery from radiation-induced hemopoietic aplasia. Female B6D2F1 mice were exposed to a sublethal 7.75-Gy dose of 60Co radiation followed by subcutaneous administration of either saline, recombinant murine (rm) MGF (100 micrograms/kg/day), rmGM-CSF (100 micrograms/kg/day), rmIL-3 (100 micrograms/kg/day), or combinations of these cytokines on days 1-17 postirradiation. Recoveries of bone marrow and splenic spleen colony-forming units (CFU-s), granulocyte macrophage colony-forming cells (GM-CFC), and peripheral white blood cells (WBC), red blood cells (RBC) and platelets (PLT) were determined on days 14 and 17 during the postirradiation recovery period. MGF administered in combination with GM-CSF or in combination with GM-CSF plus IL-3 either produced no greater response than GM-CSF alone or down-regulated the GM-CSF-induced recovery. These results sharply contrasted results of in vitro studies evaluating the effects of these cytokines on induction of GM-CFC colony formation from bone marrow cells obtained from normal or irradiated B6D2F1 mice, in which MGF synergized with GM-CSF or GM-CSF plus IL-3 to increase both GM-CFC colony numbers and colony size. These studies demonstrate a dichotomy between MGF-induced effects in vivo and in vitro and emphasize that caution should be taken in attempting to predict cytokine interactions in vivo in hemopoietically injured animals based on in vitro cytokine effects.

Haematological effects of rhGM-CSF in dogs exposed to total-body irradiation with a dose of 2.4 Gy.

It was the specific aim of this study to test the stimulatory effects of recombinant human GM-CSF (rhGM-CSF) on haemopoietic regeneration in dogs which had received total-body irradiation (TBI) with a dose of 2.4 Gy. In normal dogs rhGM-CSF given subcutaneously at 10 microgram/kg per day or 30 microgram/kg per day for 21 days caused strong but transient increases in the peripheral blood neutrophils. The monocyte counts also showed a transient rise during treatment in a dose-dependent fashion, whereas the lymphocyte counts increased only at the higher dose of rhGM-CSF and the platelet counts were transiently depressed during the course of the treatment. In the irradiated animals treatment with rhGM-CSF decreased the severity and shortened the duration of neutropenia but had no significant influence on monocyte or lymphocyte recovery. The granulocyte values showed a characteristic pattern of fluctuations with the first peak occurring at the same time (day 10 to day 13) when the abortive rise was observed in the untreated dogs. In contrast the GM-CFC in the peripheral blood remained depressed during the whole treatment course, similar to the untreated irradiated controls. These results indicate that treatment with GM-CSF can be an effective biological monotherapy for radiation-induced bone marrow failure, but that for higher radiation doses the number of GM-CSF responsive target cells will become a critical determinant of therapeutic efficacy.

Myelosuppressive effects of cytosine arabinoside (Ara-C) on growth factor-dependent human long-term bone marrow cultures (LTBMC).

Freshly isolated human mononuclear cells (5 x 10(6)) were incubated in a Dexter-type long-term bone marrow culture (LTBMC) system to study myelosuppressive effects of cytosine arabinoside (Ara-C) in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3). Differential counts (dc) of the nonadherent cell (nac) populations, starting with culture initiation, were performed weekly. After one week of simultaneous incubation of LTBMCs with either cytokine (100 ng/ml) and Ara-C (1 microgram/ml), nac numbers were markedly reduced compared to controls. Dc after week 1 of culture demonstrated significant decreases of all myeloid cell fractions except for macrophages, which remained unaffected. Growth factor-dependent LTBMCs exposed to Ara-C showed recovery of promyelocytic, metamyelocytic, and polymorphonuclear cell numbers up to control values (cultures without Ara-C exposure) in weeks 3 to 6. Intriguingly, high-proliferative, early myeloid progenitor cells (myeloblasts) appeared at high rates only in IL-3-dependent LTBMCs with and without Ara-C exposure. Nac numbers in LTBMCs exposed to Ara-C alone declined rapidly; after two weeks of culture only negligible numbers of viable nac were maintained. Plating experiments of nac in the presence of GM-CSF were performed weekly. Granulocyte-macrophage colony-forming units (CFU-GM) yields for nac from IL-3 LTBMCs were consistently higher than those for nac from GM-CSF LTBMCs. Ara-C exposure reduced CFU-GM numbers generated with nac from GM-CSF LTBMCs to 10% of GM-CSF controls (week 1). However, CFU-GM numbers grown with nac from Ara-C exposed GM-CSF-dependent LTBMCs recovered above control levels after week 3.(ABSTRACT TRUNCATED AT 250 WORDS)

An enzyme linked immunosorbent assay for the detection of human interleukin 3 in serum.

A sandwich enzyme immunoassay was developed for measuring human Interleukin 3 (IL-3) in human and animal sera. Polyclonal and monoclonal antibodies were raised against the recombinant human protein. These have been used to develop an immunoassay which can detect down to 10 pg/ml of human IL-3. The assay involves a polyclonal rabbit antibody coupled to a solid phase and a mouse monoclonal antibody-horseradish peroxidase conjugate as the detection antibody. Unlike the classical bone marrow assay and other cell line based bioassays for IL-3, the immunoassay was specific for the cytokine showing no or only negligible cross-reactivity with IL-1, IL-2, IL-6, erythropoietin, granulocyte colony-stimulating factor (G-CSF) and GM-CSF. The assay does not exhibit interfering matrix effects when used for the estimation of human IL-3 in serum samples.

Highly specific and highly sensitive enzyme immunoassays for antibodies to human interleukin 3 (IL-3) and human erythropoietin (EPO) in serum.

Two versatile, rapid, easy-to-perform and highly sensitive enzyme immunoassays (ELISAs) were developed for the detection and quantification of antibodies to human IL-3 and human EPO in serum samples from both man and laboratory animals. These one-step assays are based on the "inverse sandwich" principle, where antibodies present in the sample linked both the solid phase antigen bound to a microtiter plate and the free antigen, which had been covalently coupled to horseradish peroxidase. The limits of detection are lower than those of the neutralization bioassays; antibodies to IL-3 and to EPO were detected at concentrations as low as 2 ng/ml and 5 ng/ml, respectively. No cross-reactivity with related proteins and no interfering matrix effects were observed when used for the estimation of antibodies against rhu IL-3 and rhu EPO in serum samples of various species. Thus both assays were used without modification for the screening of antibodies in serum samples from both man and animal during treatment with these hematopoietic hormones.

Optimizing autologous blood donation by recombinant human erythropoietin (rhu-EPO) and interleukin 3 (IL-3).

The transfusion of autologous blood protects surgical patients from both the transfusion transmitted diseases (AIDS, posttransfusion hepatitis) and the immunosuppressive effects of homologous blood. Nevertheless, the use of autologous blood is still unsatisfactory, mainly because of the elaborated logistics, organization and technique required and the often insufficient amounts of autologous blood gained. Today, the major growth-factors of erythropoiesis are available as recombinant analogues. In the studies reviewed here, we investigated the effects of rhu-EPO and IL-3 on perioperative erythropoiesis in two canine models of acute isovolemic hemodilution. Different therapeutic concepts are compared with respect to preoperative changes in hematocrit, the volume of autologous blood gained and the duration of postdilutional anemia.

Bone marrow obtained during hip surgery: a novel source for studies of hemopoiesis in human long-term bone marrow culture (LTBMC).

Studies with human bone marrow cells are often impaired by the poor quality of sternal aspirates due to varying numbers of contaminating blood cells before enrichment procedures and insufficient progenitor cell yields. In this study we report on experiments performed with human bone marrow cells isolated from a) spongiose bone fragments collected during hip surgery in patients with osteoarthritis or b) sternal aspirates. After Ficoll-Histopaque density gradient centrifugation absolute cell numbers were always lower in the samples obtained from sternal aspirates. However, both sources proved to yield the same proportions of the respective myeloid cell populations. Human long-term bone marrow cultures (LTBMC), semi-solid agar assays (CFU-GM day 14), and 3H-thymidine incorporation assays proved the comparability of the two bone marrow sources.

Development of a rapid, highly sensitive, non-radioactive assay system for hematopoietic growth factors.

The aim of this study was to develop non-radioactive cell line proliferation assays. The human leukemic cell line TF1 (Kitamura et al., 1989) was used for the determination of the specific biological activity of recombinant human (rhu) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhu Interleukin 3 (IL-3) by a simple and economical fluorometric assay with a sensitivity similar to the measurement of 3H-thymidine uptake. The TF1 cell line responds to rhu IL-3, rhu GM-CSF and to a lesser extent to rhu Erythropoietin (EPO) and mast cell growth factor (MGF), but not to rhu G-CSF. It is dependent upon rhu GM-CSF for survival in culture. For the proliferation assay 1 x 10(4) TF1 cells were incubated with 20 ng - 0.256 pg rhu GM-CSF or rhu IL-3 at 37 degrees C and 5% CO2 in humidified atmosphere. After 48 h the cells were washed twice with PBS and were incubated with 4-Methylumbelliferyl-heptanoate for 60 min. Fluorescence was determined on a Titertek Fluoroskan II (Flow Lab.), and results were given as fluorescence units using a 355 nm excitation filter and a 480 nm emission filter. The developed assay showed an interassay variability lower than 15%. The sensitivity of the proliferation assays in the same range as the thymidine incorporation assays.

Studies on the efficacy of mast cell growth factor (c-kit ligand) in vitro as well as in vivo.

We tested the ability of recombinant human (rhu) mast cell growth factor (MGF), also known as c-kit ligand, to stimulate the colony formation of human bone marrow cells in semisolid medium alone and in combination with rhu erythropoietin (EPO), rhu Interleukin 3 (IL-3), rhu granulocyte colony stimulating factor (G-CSF) and rhu granulocyte-macrophage colony stimulating factor (GM-CSF). The addition of MGF to cultures containing EPO or EPO + IL-3, GM-CSF and G-CSF, resp., resulted in macroscopic erythroid burst-forming units (BFU-E). Multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]) progenitors were stimulated by MGF in the presence of EPO. Colony-forming unit granulocyte-macrophage (CFU-GM) were activated by MGF only in combination with GM-CSF. The combination of MGF with EPO was used for synergism studies in healthy cynomolgus monkeys. In the chosen concentration MGF alone had no effect on white blood cell (WBC) counts and on platelets, but a slight effect on reticulocytes. EPO by itself increased reticulocyte counts with no effects on WBC or platelets. The combination of both factors resulted in a significant increase of reticulocytes. No other effects were seen. These studies demonstrate the potent synergistic interaction of MGF and other hematopoietic growth factors.

Comparative analysis of the influences of IL-1, IL-3 and GM-CSF on the commitment of granulocyte-macrophage progenitors in vitro.

Our experiments were directed towards the detection of the influence of interleukin-1 (IL-1); interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the generation of granulocyte-macrophage progenitor cells. We also set out to examine whether this process is connected with changes within the early precursor cell compartment. Bone marrow suspension cultures (12 days) supplemented with these cytokines were tested for the presence of GM colony-forming cells (GM-CFC) in a colony-forming unit assay. The percentage of CD34+ and HLA-DR+ as well as the number of blasts and promyelocytes were estimated cytofluorometrically and morphologically. The proliferative effect of GM-CSF was associated with a net increase of GM-CFC and HLA-DR+ myeloid cells and a decrease in the percentage of CD34+ early precursor cells. IL-3 acted similarly and also caused an absolute decrease of CD34+ cells in the cultures. IL-1 did not stimulate the generation of blasts or GM-CFC but elevated the number of CD34- as well as HLA-DR-expressing cells in the cultures. These results imply that GM-CSF supported the maintenance of hematopoiesis in vitro. The transition from early precursor cells to committed myeloid progenitor cells (GM-CFC) and more mature precursor cells (G-CFC, M-CFC) may be supported by GM-CSF without affecting the self-renewing capacity of CD34+ early precursors. In contrast, the blast-generating and proliferation-inducing action of IL-3 is associated with a drop in the total number of CD34+ stem cells. An efficient renewal of this population obviously depends on the presence of IL-1.