Therapeutic efficacy of recombinant human leukemia inhibitory factor in a primate model of radiation-induced marrow aplasia

The therapeutic efficacy of recombinant human leukemia inhibitory factor (LIF) was examined in a nonhuman primate model of radiation- induced marrow aplasia. Rhesus monkeys received 450 cGy of total-body, 1:1 mixed neutron:gamma radiation. For 23 days thereafter, each monkey received a daily subcutaneous injection of LIF or human serum albumin (HSA) at a dose of 15 micrograms/kg body weight. Complete blood counts and white blood cell differentials were monitored for 60 days postirradiation. Administration of LIF significantly decreased (P < or = .05) the duration of thrombocytopenia (platelet count < 30,000 or 20,000/microL), ie, 9.3 days or 6.3 days, respectively, versus the HSA- treated control monkeys, 12.2 days or 10.2 days, respectively. Treatment with LIF did not alter the duration of neutropenia (absolute neutrophil count < 1,000/microL) as compared with the HSA-treated control monkeys. Cytokine administration did not exacerbate the radiation-induced anemia observed in the HSA-treated control monkeys.     

Combination therapy for radiation-induced bone marrow aplasia in nonhuman primates using synthokine SC-55494 and recombinant human granulocyte colony-stimulating factor

Combination cytokine therapy continues to be evaluated in an effort to stimulate multilineage hematopoietic reconstitution after bone marrow myelosuppression. This study evaluated the efficacy of combination therapy with the synthetic interleukin-3 receptor agonist, Synthokine- SC55494, and recombinant methionyl human granulocyte colony-stimulating factor (rhG-CSF) on platelet and neutrophil recovery in nonhuman primates exposed to total body 700 cGy 60Co gamma radiation. After irradiation on day (d) 0, cohorts of animals subcutaneously received single-agent protocols of either human serum albumin (HSA; every day [QD], 15 micrograms/kg/d, n = 10), Synthokine (twice daily [BID], 100, micrograms/kg/d, n = 15), rhG-CSF (QD, 10 micrograms/kg/d, n = 5), or a combination of Synthokine and rhG-CSF (BID, 100 and 10 micrograms/kg/d, respectively, n = 5) for 23 days beginning on d1. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (absolute neutrophil count < 500/microL) and thrombocytopenia (platelet count < 20,000/microL) were assessed. Animals were provided clinical support in the form of antibiotics, fresh irradiated whole blood, and fluids. All cytokine protocols significantly (P < .05) reduced the duration thrombocytopenia versus the HSA-treated animals. Only the combination protocol of Synthokine + rhG-CSF and rhG-CSF alone significantly shortened the period neutropenia (P < .05). The combined Synthokine/rhG-CSF protocol significantly improved platelet nadir versus Synthokine alone and HSA controls and neutrophil nadir versus rhG-CSF alone and HSA controls. All cytokine protocols decreased the time to recovery to preirradiation neutrophil and platelet values. The Synthokine/rhG-CSF protocol also reduced the transfusion requirements per treatment group to 0 among 5 animals as compared with 2 among 5 animals for Synthokine alone, 8 among 5 animals for rhG-CSF, and 17 among 10 animals for HSA. These data showed that the combination of Synthokine, SC-55494, and rhG-CSF further decreased the cytopenic periods and nadirs for both platelets and neutrophils relative to Synthokine and rhG-CSF monotherapy and suggest that this combination therapy would be effective against both neutropenia and thrombocytopenia consequent to drug- or radiation- induced myelosuppression.     

Enhanced marrow recovery by short preincubation of marrow allografts with human recombinant interleukin-3 and granulocyte-macrophage colony-stimulating factor.

We studied an alternative method of using hematopoietic growth factors (HGFs) to enhance hematopoietic recovery in patients undergoing bone marrow transplantation (BMT), by short in vitro preincubation. Twenty consecutive patients with leukemia received T-cell-depleted allografts using Campath-1G. Two thirds of the marrow was infused on the scheduled day of transplant and one third of the marrow following preincubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) on day 4. Engraftment parameters and duration of hospitalization were compared by actuarial analysis to those of 40 historical controls. Patients receiving the incubated boost had significantly faster platelet recovery (P = .017) and shorter hospitalization period (P = .001) when compared with the control subjects. Platelet count reached greater than 25 x 10(9)/L on day 17 (median) in the study group and on day 23 in the controls. The median duration of hospitalization was 20 and 36 days, respectively. In the early posttransplantation follow-up, two of four patients in the study group died as a result of graft rejection, while all 13 deaths in the control group resulted from complications associated with marrow suppression. We suggest that pretransplant in vitro activation of bone marrow cells with IL-3 and GM-CSF may prove to be an efficient method for enhancing marrow recovery after BMT.     

Activities of granulocyte-macrophage colony-stimulating factor and interleukin-3 on monocytes

We examined the actions of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) on human monocytes, using a serum-free culture system. GM-CSF and IL-3 did not promote the differentiation of monocytes into macrophages but rather into cells with a phenotype compatible with that of immature dendritic cells (DCs). The addition of fetal bovine serum to serum-free cultures with GM-CSF or IL-3 restored the differentiation of monocytes into macrophages. Cells generated with GM-CSF or IL-3 elicited phagocytic activity. Cells generated in the presence of GM-CSF or IL-3, followed by the addition of tumor necrosis factor-alpha, displayed a phenotype of mature DCs, and primed and stimulated immunogenic peptide-specific T lymphocytes. Surprisingly, GM-CSF and IL-3 inhibited macrophage colony-stimulating factor (M-CSF)-dependent differentiation of monocytes into macrophages and induced differentiation into immature DCs. We asked if the inhibition of M-CSF-dependent differentiation into macrophages by GM-CSF or IL-3 was associated with the expression of M-CSF receptors (M-CSFR). GM-CSF or IL-3 down-regulated the expression of M-CSFR. These data demonstrate that GM-CSF and IL-3 primarily support the differentiation of monocytes into DCs and inhibit M-CSF-dependent differentiation into macrophages by suppressing the expression of M-CSFR, thereby promoting differentiation into DCs.     

Enhancement of the grafting efficiency of transplanted marrow cells by preincubation with interleukin-3 and granulocyte-macrophage colony-stimulating factor

To improve the grafting efficiency of transplanted murine hematopoietic progenitors, we briefly preincubated mouse bone marrow cells with interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) ex vivo before their transplantation into irradiated recipients. This treatment was translated into an increase in the seeding efficiency of colony-forming unit-spleen (CFU-S) and CFU-GM after transplantation. Not only was the concentration of CFU-S in the tibia increased 2 and 24 hours after transplantation, but the total cell number and CFU-S and CFU-GM concentrations were persistently higher in IL-3- and GM-CSF-treated groups 1 to 3 weeks after transplantation. In addition, the survival of animals as a function of transplanted cell number was persistently higher in IL-3- and GM-CSF-treated groups compared with controls. The data indicate that the pretreatment of marrow cells with IL-3 and GM-CSF before transplantation increases the seeding efficiency of hematopoietic stem cells and probably other progenitor cells after transplantation. This increased efficiency may be mediated by upward modulation of homing receptors. Therefore, ex vivo preincubation of donor marrow cells with IL-3 and GM-CSF may be a useful tactic in bone marrow transplantation.     

Effect of N-methionine-free, bacterially synthesized recombinant human granulocyte-macrophage colony-stimulating factor in a primate model

We demonstrate the in vivo effects of bacterially synthesized, N-methionine-free recombinant human granulocyte-macrophage colony stimulating factor (rh GM-CSF) using a crab-eating monkey model. Monkeys were treated with cyclophosphamide (60 mg/kg) and administered with rh GM-CSF (30 micrograms/kg/d) subcutaneously (s.c.) for 7 days. Within 12 h, a transient increase of neutrophils (greater than 15.0 x 10(9)/l) was observed, and complete recovery of WBC counts was obtained by d 9 (d 16 in control monkeys). Neutrophils and eosinophils were absolutely increased (greater than 8 x 10(9)/l) on d 10. Readministration of rh GM-CSF (30 micrograms/kg/d, s.c.) for 3 d (including control monkeys) revealed absolute increases of neutrophils, eosinophils, monocytes and platelets. A two-fold increase of granulocyte/macrophage colony-forming units was also seen in the bone marrow, while the number of burst-forming units-erythroid was not affected. These data indicate that rh GM-CSF of this type stimulates granulopoiesis and thrombopoiesis in vivo.     

Therapeutic efficacy of recombinant interleukin-6 (IL-6) alone and combined with recombinant human IL-3 in a nonhuman primate model of high-dose, sublethal radiation-induced marrow aplasia

Using a nonhuman-primate model of radiation-induced bone marrow aplasia, we examined whether the single, concomitant, or sequential administration of recombinant human interleukin-3 (IL-3) and IL-6 would promote bone marrow regeneration measured by an increase in circulating platelets (PLT) and neutrophils (PMN). Rhesus monkeys were irradiated at 450 cGy and were randomly assigned to one of five treatment protocols, receiving IL-6; IL-3; combined IL-6 and IL-3; sequential IL- 3 and IL-6; or human serum albumin (HSA) as a control. Cytokines or HSA were administered at total dosages of 15 micrograms/kg/day. Complete blood counts and white blood cell differentials were monitored for 60 days postirradiation. Both IL-3 and IL-6 significantly enhanced the regeneration of PLTs and decreased the duration of thrombocytopenia (P = .005) without affecting PMN recovery. The radiation-induced anemia that was observed in the HSA-treated controls was less severe and resolved more quickly in the IL-6 treated animals. Sequential IL-3/IL-6 significantly increased the production of PLTs when compared with the HSA-treated controls (P = .003) and monkeys receiving concomitant IL- 3/IL-6 (P = .041) but did not alter PMN levels significantly (P = .80). Coadministration of IL-6 and IL-3 did not enhance PLT but improved PMN recovery over IL-6 alone. In this primate model of marrow aplasia, IL-6 significantly enhanced the regeneration of PLTs but had no significant effect on PMN production, and did not exacerbate radiation-induced anemia. Furthermore, the use of sequentially administered IL-3 and IL-6 may improve PLT recovery as compared with concurrent IL-3/IL-6 administration, although this protocol is not significantly different in effect than either cytokine alone.     

Effects of interleukin-3 and granulocyte-macrophage colony-stimulating factor on thrombopoiesis in congenital amegakaryocytic thrombocytopenia

Amegakaryocytic thrombocytopenia (AMT) is a rare and often fatal disorder of infancy and childhood presenting with isolated thrombocytopenia that progresses to marrow failure. The defect in thrombopoiesis is not well understood nor is the etiology of the progressive marrow failure. No standard modality of treatment exists. Here, we evaluated the capacity of marrow cells isolated from five patients with AMT and progressive marrow failure to generate megakaryocyte progenitor cells (CFU-MK). These in vitro studies demonstrated assayable numbers of CFU-MK from all patient bone marrows that responded in vitro to the addition of interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or the combination of both. These findings suggest that the defect in AMT might be partially correctable by the administration of these cytokines. A Phase I/II trial of in vivo administration of these same hematopoietins in the identical patients was conducted in which no significant toxicity was observed. IL-3 but not GM-CSF administration resulted in improved platelet counts in two patients and decreased bleeding and transfusion requirement in the remaining three. No clinical benefit was observed when GM-CSF was administered after IL-3 pretreatment. Prolonged IL-3 administration has resulted in platelet increases in an additional two patients. In vitro responsiveness of CFU- MK to either cytokine did not predict the degree of clinical response. Although the optimal dose and schedule of IL-3 either alone or in combination remains to be established, this study suggests that IL-3 may contribute to the treatment of patients with AMT.     

Biological properties in vitro of a combination of recombinant murine interleukin-3 and granulocyte-macrophage colony-stimulating factor

The effect of recombinant murine interleukin-3 (rIL-3) and recombinant murine granulocyte-macrophage colony-stimulating factor (rGM-CSF) on in vitro murine myeloid progenitor cell (CFU-C) growth and on the function of murine resident peritoneal macrophages was investigated. Both rIL-3 and rGM-CSF are known to support the growth of CFU-C and, when combined, were found to act synergistically to induce the development of an increased number of CFU-C. The distribution pattern of myeloid colonies in the presence of these two growth factors was in general similar to that in the presence of rGM-CSF alone. Both rGM-CSF and rIL-3 enhanced the phagocytosis of Candida albicans (CA) by mature macrophages producing an increase in the percentage of phagocytosing cells as well as an increase in the number of yeast particles ingested per cell. No additive effect on the phagocytosis was observed when the two growth factors were added concurrently. rGM-CSF, but not rIL-3, enhanced the killing of CA by macrophages. This killing was inhibited by scavengers of oxygen radicals.     

Effect of granulocyte-macrophage colony-stimulating factor and interleukin-3 on interleukin-8 production by human neutrophils and monocytes

Interleukin-8 (IL-8) is a major neutrophil chemoattractant and functional stimulant that is induced by IL-1, tumor necrosis factor alpha (TNF alpha), and lipopolysaccharide (LPS). We report that recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhIL-3 are also potent inducers of IL-8 messenger RNA (mRNA) accumulation and protein secretion by normal peripheral blood monocytes. Neutrophils produce IL-8 in response to GM-CSF but not to IL- 3. In contrast, recombinant human granulocyte-CSF (rhG-CSF), at concentrations as high as 100 ng/mL, does not induce IL-8 in either cell type. rhGM-CSF also induces IL-8 mRNA expression and IL-8 protein in the promonocytic cell line, U-937, whereas rhG-CSF does not. IL-8 secretion by monocytes was stimulated within 2 hours after incubation with rhGM-CSF or rhIL-3. Stimulation of neutrophils with rhGM-CSF resulted in an increase in cell-associated IL-8 at 4 hours. At 24 hours, cell-associated IL-8 levels declined, whereas secreted IL-8 levels increased. In contrast, virtually all IL-8 induced in monocytes appeared as secreted protein. Neither rhGM-CSF nor rhIL-3 induced detectable secretion of IL-1, TNF alpha, or IL-6 protein by monocytes. rhGM-CSF, and to a lesser degree rhIL-3, potently stimulated IL-8 secretion in cultures of heparinized whole blood, whereas rhG-CSF had no significant effect on IL-8 secretion. Induction of IL-8 by GM-CSF may be physiologically important in enhancing the acute inflammatory response.     

Modulation and induction of eosinophil chemotaxis by granulocyte-macrophage colony-stimulating factor and interleukin-3.

Eosinophilia and eosinophil function are regulated by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5. We have investigated the modulatory role of GM-CSF and IL-3 on the platelet-activating factor (PAF)-, neutrophil-activating factor (NAF/IL-8)-, leukotriene B4 (LTB4)-, N-formyl-methionyl-leucyl-phenylalanine (FMLP)-, and human complement factor C5a-induced chemotaxis of eosinophils from normal individuals. These eosinophils show a chemotactic response toward PAF, LTB4, and C5a, but not to NAF/IL-8 and FMLP. Preincubation of the eosinophils with picomolar concentrations of GM-CSF caused a significant increase in the response toward LTB4 and induced a significant chemotactic response toward NAF/IL-8 and FMLP. Preincubation of the eosinophils with picomolar concentrations of IL-3 also induced a chemotactic response toward NAF/IL-8 and FMLP, and enhanced the PAF-induced chemotaxis response toward C5a was not influenced by both cytokines. Nanomolar concentrations of GM-CSF or IL-3 caused a significant inhibition of the C5a-induced chemotaxis. The LTB4-induced chemotaxis was also significantly inhibited in case of GM-CSF. At these concentrations both GM-CSF and IL-3 acted as chemotaxins for eosinophils were washed after pretreatment with GM-CSF and IL-3 the potentiation of the chemotactic response remained, whereas the inhibitory mode of action disappeared. Our data indicate that at picomolar concentrations the cytokines GM-CSF and IL-3 can modulate eosinophil chemotaxis and at nanomolar concentrations these cytokines can act as chemotaxins for eosinophils.     

Overlapping roles for granulocyte-macrophage colony-stimulating factor and interleukin-3 in eosinophil homeostasis and contact hypersensitivity

Studies of mice rendered deficient in granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) have established unique roles for these cytokines in pulmonary homeostasis, resistance to infection, and antigen-specific T- and B-cell responses. In addition to these distinctive properties, however, GM-CSF and IL-3 also stimulate the development and activation of hematopoietic cells in many similar ways, raising the possibility that each factor might partially compensate for the other's absence in singly deficient mice. To test whether endogenous GM-CSF and IL-3 mediate redundant functions in vivo, we generated mice lacking both cytokines through sequential gene targeting experiments in embryonic stem (ES) cells. Surprisingly, doubly deficient animals, but not single knockouts, showed increased numbers of circulating eosinophils. Doubly deficient mice, moreover, developed weaker contact hypersensitivity reactions to haptens applied epicutaneously than mice deficient in either factor alone. Together, these findings delineate overlapping roles for GM-CSF and IL-3 in hematopoiesis and immunity. (Blood. 2001;97:922-928)     

Polycythemia vera. II. Hypersensitivity of bone marrow erythroid, granulocyte-macrophage, and megakaryocyte progenitor cells to interleukin-3 and granulocyte-macrophage colony-stimulating factor.

Polycythemia vera (PV) is a clonal disease of the hematopoietic stem cell characterized by a hyperplasia of marrow erythropoiesis, granulocytopoiesis, and megakaryocytopoiesis. We previously reported that highly purified PV blood burst-forming units-erythroid (BFU-E) are hypersensitive to recombinant human interleukin-3 (rIL-3). Because these cells may be only a subset, and not representative of marrow progenitors, we have now studied partially purified marrow hematopoietic progenitor cells. Dose-response experiments with PV marrow BFU-E showed a 38-fold increase in sensitivity to rIL-3 and a 4.3-fold increase in sensitivity to recombinant human erythropoietin (rEpo) compared with normal marrow BFU-E. In addition, PV marrow colony-forming units-granulocyte-macrophage (CFU-GM) and CFU-megakaryocyte (CFU-MK) also showed a marked hypersensitivity to rIL-3 and to human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Dose-response curves with rGM-CSF and blood BFU-E showed a 48-fold increase in sensitivity. No effect of rIL-4, rIL-6, human recombinant granulocyte-CSF (rG-CSF), or macrophage-CSF (rM-CSF) was evident, nor was there any effect of PV cell-conditioned medium on normal BFU-E, when compared with normal cell-conditioned medium. Autoradiography with 125I-rEpo showed an increase in Epo receptors after maturation of PV BFU-E to CFU-E similar to that shown with normal BFU-E, but no increase of specific binding of 125I-rIL-3 by PV CD34+ cells was seen compared with normal CD34+ cells. These studies show that PV marrow hematopoietic progenitor cells are hypersensitive to rIL-3 and rGM-CSF, similar to PV blood BFU-E. While the mechanism does not appear to be due to enhanced binding of rIL-3, the hypersensitivity of PV progenitor cells to IL-3 and GM-CSF may be a key factor in the pathogenesis of PV.     

Dependence of interleukin-1-induced arthritis on granulocyte-macrophage colony-stimulating factor

OBJECTIVE: To determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage CSF (M-CSF or CSF-1) are involved in the methylated bovine serum albumin/interleukin-1 (mBSA/IL-1)-induced arthritis model. METHODS: Following systemic injection, IL-1 has been shown to augment a weak inflammatory response to mBSA in murine joints and to induce an acute erosive arthritis. GM-CSF and M-CSF have been implicated in inflammatory reactions, including those in joints, and have recently been shown to exacerbate murine arthritis. Since in vitro studies have found that IL-1 can enhance GM-CSF and M-CSF production, we reasoned that they might be playing a part in IL-1-mediated arthritis. GM-CSF-deficient (GM-CSF-/-) and M-CSF-deficient (op/op) mice were injected intraarticularly with mBSA and subcutaneously with IL-1. Arthritis was monitored histologically on day 7. Normal mice were also treated intraperitoneally with blocking monoclonal antibodies to GM-CSF and M-CSF, and to the M-CSF receptor. Numbers of macrophages (Mac-2 and F4/80 staining) were monitored, as was the number of cycling (bromodeoxyuridine-positive) cells. RESULTS: GM-CSF-/- mice and normal mice treated with anti-GM-CSF antibody did not show IL-1-induced arthritis progression. There was a dramatic reduction in synovial cellularity, including reduced numbers of macrophages and cycling cells. The op/op mice did not develop mBSA/IL-1-induced disease, but blocking antibody to M-CSF or to the M-CSF receptor failed to diminish disease in normal mice. CONCLUSION: GM-CSF is involved in the IL-1-induced arthritis that follows mBSA injection; M-CSF involvement in the model is also suggested, since op/op mice did not develop arthritis. These studies provide the first in vivo evidence for a role of GM-CSF, and possibly M-CSF, in the proinflammatory actions of IL-1.     

Interaction of granulocyte-macrophage colony-stimulating factor and interleukin 3 in human long-term bone marrow culture.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), or a combination of both growth factors were added weekly to normal human long-term bone marrow cultures (LTBMC). GM-CSF had a greater effect on the total nonadherent cell population than the committed progenitor cells (granulocyte-macrophage colony-forming units, CFUgm), whereas IL-3 had the opposite effect and stimulated the expansion of greater numbers of CFUgm than GM-CSF. The combination of both factors had an additive effect on CFUgm. The longevity of the growth factor-treated cultures was not reduced. These data indicate that IL-3 stimulates an earlier progenitor cell population than GM-CSF and that a combination of the two factors should be more effective in vivo and could be applied to the expansion of bone marrow progenitor cells in culture before bone marrow transplantation.     

Acceleration of hematopoietic reconstitution with a synthetic cytokine (SC-55494) after radiation-induced bone marrow aplasia

The synthetic cytokine (Synthokine) SC-55494 is a high-affinity interleukin-3 (IL-3) receptor ligand that stimulates greater in vitro multilineage hematopoietic activity than native IL-3, while inducing no significant increase in inflammatory activity relative to native IL-3. The aim of this study was to investigate the in vivo hematopoietic response of rhesus monkeys receiving Synthokine after radiation-induced marrow aplasia. Administration schedule and dose of Synthokine were evaluated. All animals were total-body irradiated (TBI) with 700 cGy 60Co gamma radiation on day 0. Beginning on day 1, cohorts of animals (n = 5) received Synthokine subcutaneously (SC) twice daily with 25 micrograms/kg/d or 100 micrograms/kg/d for 23 days or 100 micrograms/kg/d for 14 days. Control animals (n = 9) received human serum albumin SC once daily at 15 micrograms/kg/d for 23 days. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (NEUT; absolute neutrophil count [ANC] < 500/microL) and thrombocytopenia (THROM; platelet count < 20,000/microL) were assessed. Synthokine significantly (P < .05) reduced the duration of THROM versus the HSA-treated animals regardless of dose or protocol length. The most striking reduction was obtained in the animals receiving 100 micrograms/kg/d for 23 days (THROM = 3.5 v 12.5 days in HSA control animals). Although the duration of NEUT was not significantly altered, the depth of the nadir was significantly lessened in all animal cohorts treated with Synthokine regardless of dose versus schedule length. Bone marrow progenitor cell cultures indicated a beneficial effect of Synthokine on the recovery of granulocyte-macrophage colony-forming units that was significantly higher at day 24 post-TBI in both cohorts treated at 25 and 100 micrograms/kg/d for 23 days relative to the control animals. Plasma pharmacokinetic parameters were evaluated in both normal and irradiated animals. Pharmacokinetic analysis performed in irradiated animals after 1 week of treatment suggests an effect of repetitive Synthokine schedule and/or TBI on distribution and/or elimination of Synthokine. These data show that the Synthokine, SC55 94, administered therapeutically post-TBI, significantly enhanced platelet recovery and modulated neutrophil nadir and may be clinically useful in the treatment of the myeloablated host.     

Simultaneous antagonism of interleukin-5, granulocyte-macrophage colony-stimulating factor, and interleukin-3 stimulation of human eosinophils by targetting the common cytokine binding site of their receptors.

Human interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 are eosinophilopoietic cytokines implicated in allergy in general and in the inflammation of the airways specifically as seen in asthma. All 3 cytokines function through cell surface receptors that comprise a ligand-specific alpha chain and a shared subunit (beta(c)). Although binding of IL-5, GM-CSF, and IL-3 to their respective receptor alpha chains is the first step in receptor activation, it is the recruitment of beta(c) that allows high-affinity binding and signal transduction to proceed. Thus, beta(c) is a valid yet untested target for antiasthma drugs with the added advantage of potentially allowing antagonism of all 3 eosinophil-acting cytokines with a single compound. We show here the first development of such an agent in the form of a monoclonal antibody (MoAb), BION-1, raised against the isolated membrane proximal domain of beta(c). BION-1 blocked eosinophil production, survival, and activation stimulated by IL-5 as well as by GM-CSF and IL-3. Studies of the mechanism of this antagonism showed that BION-1 prevented the high-affinity binding of (125)I-IL-5, (125)I-GM-CSF, and (125)I-IL-3 to purified human eosinophils and that it bound to the major cytokine binding site of beta(c). Interestingly, epitope analysis using several beta(c) mutants showed that BION-1 interacted with residues different from those used by IL-5, GM-CSF, and IL-3. Furthermore, coimmunoprecipitation experiments showed that BION-1 prevented ligand-induced receptor dimerization and phosphorylation of beta(c), suggesting that ligand contact with beta(c) is a prerequisite for recruitment of beta(c), receptor dimerization, and consequent activation. These results demonstrate the feasibility of simultaneously inhibiting IL-5, GM-CSF, and IL-3 function with a single agent and that BION-1 represents a new tool and lead compound with which to identify and generate further agents for the treatment of eosinophil-dependent diseases such as asthma.     

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)