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.     

Increased respiratory burst activity of neutrophils in patients with aplastic anemia: effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

The superoxide (O2-)-releasing capacity in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) and the priming effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on FMLP-induced O2-release were investigated in neutrophils from 13 patients with aplastic anemia (AA). The O2(-)-releasing capacity of AA neutrophils (0.85 +/- 0.36 nmol/5 min/1 x 10(5) cells, n = 13) was significantly (p < 0.01) increased as compared with that of normal neutrophils (0.24 +/- 0.12 nmol/5 min/1 x 10(5) cells, n = 17). There was no close relationship between the O2(-)-releasing capacity and the peripheral blood neutrophil count or the plasma concentration of C-reactive protein. The plasma concentrations of G-CSF and GM-CSF were not elevated to the detectable levels (< 0.1 ng/ml and < 0.2 ng/ml, respectively) in all patients tested. FMLP-induced O2(-)-release was further enhanced by pretreatment of cells with rhG-CSF or rhGM-CSF for 10 min at 37 degrees C, except that no significant priming by rhG-CSF was observed in five patients. The priming effect of rhGM-CSF was consistently greater than that of rhG-CSF in all patients. The i.v. administration of rhGM-CSF (6 micrograms/kg body weight/day) to one patient resulted in an increase in neutrophil O2(-)-release stimulated by FMLP. These findings indicate that neutrophils from AA patients are already primed in vivo for enhanced release of O2- and that these neutrophil functions are further potentiated by rhG-CSF or rhGM-CSF.     

Antagonistic effects of interleukin 6 and G-CSF in the later stage of human granulopoiesis in vitro.

The effect of recombinant human interleukin 6 (rhIL-6) on the in vitro growth of human bone marrow myeloid progenitors (granulocyte-macrophage colony-forming units, CFU-GM) was investigated. Recombinant human IL-6 by itself did not induce colony formation. When rhIL-6 at various concentrations was added to the CFU-GM colony cultures containing recombinant human granulocyte colony-stimulating factor (rhG-CSF) or recombinant human granulocyte-monocyte/macrophage colony-stimulating factor (rhGM-CSF), rhIL-6 significantly suppressed the colony formation induced by rhG-CSF, but not by rhGM-CSF. This suppressive effect of rhIL-6 on rhG-CSF-induced, but not rhGM-CSF-induced colony formation was confirmed by using an MY10(+)-cell-enriched population. Neither interleukin 3 nor interleukin 1 alpha suppressed the growth of myeloid progenitors. The preincubation of bone marrow cells with rhIL-6 for a short time (30 min) resulted in a reduction of colonies induced by rhG-CSF, but not by rhGM-CSF. The suppressive effect of rhIL-6 on rhG-CSF-induced colony formation was not observed when the cells were preincubated together with rhG-CSF at a high ratio of rhG-CSF to rhIL-6. The rhIL-6-mediated suppressive effect was further confirmed by blocking the effect by the anti-IL-6 antibody. These results suggest antagonistic interaction between IL-6 and G-CSF in the later differentiation of myeloid progenitors.     

Monocyte-mediated killing of human leukaemia is enhanced by administration of granulocyte-macrophage colony stimulating factor following chemotherapy

We studied the capacity of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) to modulate monocyte anti-leukaemic activity when administered to patients following myelosuppressive chemotherapy. The leukaemic cell lines K562, U937 and KG-1 were used as models of human leukaemia as they exhibit differential sensitivity to cell-mediated or TNF-mediated cytotoxicity. Monocyte tumouricidal activity was augmented by rhGM-CSF or lipopolysaccharide (LPS) alone in vitro against leukaemic blasts, whereas granulocyte-colony stimulating factor (rhG-CSF) was without effect. rhGM-CSF and LPS exhibited an additive effect in stimulating the cytotoxic effect of monocytes against K562 blasts compared with either agent alone ( P  < 0.001). Both cell-mediated and soluble TNF-mediated killing of leukaemic blasts was augmented by rhGM-CSF administration to patients following chemotherapy. This effect persisted for up to 4 weeks after cessation of GM-CSF therapy. The administration of rhGM-CSF significantly increased the anti-leukaemic activity of monocytes against leukaemic targets that were resistant to secreted TNF, probably via a transmembrane TNF-dependent mechanism. Therapy with rhG-CSF exhibited a minimal effect. We conclude that administration of rhGM-CSF, but not rhG-CSF, augments the tumouricidal properties of the monocyte-macrophage system, particularly during recovery from myelosuppressive chemotherapy. Moreover, the killing mechanism is direct and not mediated by an antibody-dependent cellular cytotoxic (ADCC) mechanism. Killing of TNF-resistant leukaemic cells in particular may be augmented via cell-to-cell contact.     

Various human hematopoietic growth factors (interleukin-3, GM-CSF, G-CSF) stimulate clonal growth of nonhematopoietic tumor cells

We have studied the effect of recombinant human hematopoietic growth factors (interleukin-3 [rhIL-3], granulocyte-macrophage colony-stimulating factor [rhGM-CSF], and granulocyte CSF [rhG-CSF]) on the clonal growth of human colon adenocarcinoma cell lines HTB-38, CCL 187, and WiDr (CCL 218). The factors stimulated clonal growth of HTB-38 and CCL 187 in a capillary modification of the human tumor clonogenic assay in agar up to twofold. There were dose-response correlations over a range of 1 to 10,000 U/mL for rhIL-3, rhGM-CSF, and rhG-CSF. Incubation with neutralizing monoclonal antibodies abolished the stimulation of clonal growth by rhGM-CSF. The WiDr cell line was nonresponsive to rhIL-3 and rhGM-CSF. These results represent the first evidence that a variety of hematopoietic growth factors can stimulate the growth of clonogenic cells of some nonhematopoietic malignant cell lines in vitro.     

Activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate oxidase and phospholipase A(2) are dissociated by inhibitors of the kinases p42(ERK2) and p38(SAPK) and by methyl arachidonyl fluorophosphonate, the dual inhibitor of cytosolic and calcium-independent phospholipase A(2)

Arachidonic acid (AA) generated by phospholipase A(2) (PLA(2)) is thought to be an essential cofactor for phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Both enzymes are simultaneously primed by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha). The possibility that either unprimed or cytokine-primed responses of PLA(2) or NADPH oxidase to the chemotactic agents formyl-methionyl-leucyl-phenylalanine (FMLP) and complement factor 5a (C5a) could be differentially inhibited by inhibitors of the mitogen-activated protein (MAP) kinase family members p42(ERK2) (PD98059) and p38(SAPK) (SB203580) was investigated. PD98059 inhibited the activation of p42(ERK2) by GM-CSF, TNF-alpha, and FMLP, but it did not inhibit FMLP-stimulated superoxide production in either unprimed or primed neutrophils. There was no significant arachidonate release from unprimed neutrophils stimulated by FMLP, and arachidonate release stimulated by calcium ionophore A23187 was not inhibited by PD98059. In contrast, PD98059 inhibited both TNF-alpha- and GM-CSF-primed PLA(2) responses stimulated by FMLP. On the other hand, SB203580 inhibited FMLP-superoxide responses in unprimed as well as TNF-alpha- and GM-CSF-primed neutrophils, but failed to inhibit TNF-alpha- and GM-CSF-primed PLA(2) responses stimulated by FMLP, and additionally enhanced A23187-stimulated arachidonate release, showing that priming and activation of PLA(2) and NADPH oxidase are differentially dependent on both the p38(SAPK) and p42(ERK2) pathways. Studies using C5a as an agonist gave similar results and confirmed the findings with FMLP. In addition, methyl arachidonyl fluorophosphonate (MAFP), the dual inhibitor of c and iPLA(2) enzymes, failed to inhibit superoxide production in primed cells at concentrations that inhibited arachidonate release. These data demonstrate that NADPH oxidase activity can be dissociated from AA generation and indicate a more complex role for arachidonate in neutrophil superoxide production.     

Suppressive effect of granulocyte-macrophage colony-stimulating factor on the generation of natural killer cells in vitro.

We investigated the effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant human granulocyte-CSF (rhG-CSF) on the generation of natural killer (NK) cells in vitro. NK cells were cultured from selected human bone marrow cells obtained after the elimination of mature T and NK cells. rhGM-CSF significantly suppressed the generation of CD56+ cells and NK activity (P less than .01) in a dose-dependent manner. The generation of large granular lymphocytes (LGL) was also suppressed in the presence of rhGM-CSF (P less than .01). In contrast, rhG-CSF had no effect on LGL (P greater than .05). Both rhGM-CSF and rhG-CSF had no influence on the CD56+ cell count in the peripheral blood. These results suggest that rhGM-CSF suppresses the in vitro generation of NK cells.     

A comparison of treatment of canine cyclic hematopoiesis with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF interleukin-3, and canine G-CSF.

Cyclic hematopoiesis in gray collie dogs is a stem cell disease in which abnormal regulation of cell production in the bone marrow causes cyclic fluctuations of blood cell counts. In vitro studies demonstrated that recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and granulocyte colony stimulating factor (G-CSF) all stimulated increases in colony formation by canine bone marrow progenitor cells. Based on these results, gray collie dogs were then treated with recombinant human (rh) GM-CSF, IL-3, or G-CSF subcutaneously to test the hypothesis that pharmacologic doses of one of these hematopoietic growth factors could alter cyclic production of cells. When recombinant canine G-CSF became available, it was tested over a range of doses. In vivo rhIL-3 had no effect on the recurrent neutropenia but was associated with eosinophilia, rhGM-CSF caused neutrophilia and eosinophilia but cycling of hematopoiesis persisted. However, rhG-CSF caused neutrophilia, prevented the recurrent neutropenia and, in the two animals not developing antibodies to rhG-CSF, obliterated periodic fluctuation of monocyte, eosinophil, reticulocyte, and platelet counts. Recombinant canine G-CSF increased the nadir neutrophil counts and amplitude of fluctuations at low doses (1 micrograms/kg/d) and eliminated all cycling of cell counts at high doses (5 and 10 micrograms/kg/d). These data suggest significant differences in the actions of these growth factors and imply a critical role for G-CSF in the homeostatic regulation of hematopoiesis.     

Antibody to Mol abrogates the increase in neutrophil phagocytosis and degranulation induced by granulocyte-macrophage colony-stimulating factor

We studied the ability of the human hemopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) to activate polymorphonuclear neutrophils (PMN) for increased phagocytosis of opsonized Candida albicans and enhanced degranulation. Exposure of neutrophils to these two growth factors resulted in an increased number of Candida phagocytosed. Pretreatment of the neutrophils with the monoclonal antibody anti-Mol abrogated the enhanced phagocytosis associated with GM-CSF priming but not that of G-CSF primed PMN. In examining the effect of these two colony-stimulating factors (CSFs) on neutrophil degranulation we found that GM-CSF induced enhanced release of lysozyme from cytochalasin-treated PMN in the presence of Candida; however, G-CSF did not. The effect of GM-CSF on lysozyme release was abrogated by anti-Mol antibody. These data suggest that GM-CSF and G-CSF prime PMN for certain enhanced functional activities by distinct mechanisms. The differential effect of the CSFs on neutrophil degranulation may relate to the more common inflammatory symptoms seen when GM-CSF is used clinically as compared to the experience with G-CSF.     

Enhancement of neutrophil function by granulocyte-macrophage colony- stimulating factor involves recruitment of a less responsive subpopulation

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances numerous functions of mature neutrophils (PMN) including phagocytosis, superoxide responses to chemotaxins, antibody-dependent cellular cytotoxicity, and expression of complement receptors. A central question concerns whether the mechanism of enhancement involves quantitative increases in the response of all cells v subpopulation recruitment. The effects of GM-CSF on individual cell light scatter changes, membrane potential, and oxidant responses induced by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP) were assessed by flow cytometry and by scoring individual cells for nitroblue tetrazolium dye (NBT) reduction. GM-CSF produced a dose- and time-dependent shift in forward light scatter that was very similar in character to that seen with FMLP or leukotriene B4 stimulation. Although not capable of depolarizing the cells directly, GM-CSF primed PMNs for enhanced membrane potential responses to FMLP by significantly increasing the proportion of depolarizing cells when compared with diluent-treated controls after a 60-minute incubation at 37 degrees C (79.4% +/- 3.4% v 29.5% +/- 4.7% GM-CSF v diluent, mean +/- SE, P less than .005, n = 11). Subpopulation recruitment by GM-CSF treatment was also demonstrated by the FMLP-elicited NBT test. Taken together, these results indicate that GM-CSF can modulate the function of mature PMN by enhancing the proportion of responsive cells.     

Treatment of severe neutropenia due to Felty's syndrome or systemic lupus erythematosus with granulocyte colony-stimulating factor.

OBJECTIVES: To examine the efficacy and safety of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for the treatment of severe neutropenia due to Felty's syndrome (FS) or systemic lupus erythematosus (SLE). METHODS: Eight patients with absolute neutrophil counts (ANC) below 1,000/microL attributable to FS (n = 4) or SLE (n = 4) were treated with rhG-CSF. The hematologic and clinical response as well as side effects were recorded. In addition, reports on the use of rhG-CSF/rhGM-CSF in FS and SLE retrieved from the English language literature were analyzed. RESULTS: RhG-CSF effectively corrected neutropenia due to FS and SLE in seven of the current eight patients. In 54 of 55 FS and SLE patients retrieved from the literature, G-CSF or GM-CSF, respectively, proved to be effective at elevating the neutrophil count, which was often associated with improvement of infectious complications. The neutrophil count often declined again when growth factor treatment was stopped but generally stabilized at a level that exceeded the pretreatment count. Side effects included rare cases of thrombocytopenia, arthralgias, and development of cutaneous leukocytoclastic vasculitis. Side effects were dose dependent and resolved when treatment was discontinued. One of our own patients and 17 previously reported patients continued to benefit from long-term administration of rhG-CSF over periods of more than 40 months. CONCLUSIONS: RhG-CSF is an effective and generally well-tolerated treatment for neutropenia due to FS or SLE. Exacerbation of the underlying rheumatic condition due to G-CSF appears to be rare if G-CSF is administered at the lowest dose effective at elevating the ANC above 1,000/microL.     

Regulation of polymorphonuclear leukocyte degranulation and oxidant production by ceramide through inhibition of phospholipase D

Exogenous C(2)-ceramide has been shown to inhibit polymorphonuclear leukocyte (PMN) phagocytosis through inhibition of phospholipase D (PLD) and downstream events, including activation of extracellular signal-regulated kinases 1 and 2, leading to the hyphothesis that the sphingomyelinase pathway is involved in termination of phagocytosis. Here it is postulated that increased PLD activity generating phosphatidic acid and diacylglycerol (DAG) is essential for superoxide release and degranulation and that ceramide, previously shown to be generated during PMN activation, inhibits PLD activation, thereby leading to inhibition of PMN function. When PMNs were primed with granulocyte colony-stimulating factor (G-CSF) and then activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP), C(2)-ceramide (10 microM) completely inhibited release of superoxide, lactoferrin, and gelatinase; the DAG analog sn-1,2-didecanoylglycerol (DiC10) (10 microM) restored oxidase activation and degranulation in the ceramide-treated cells. Similarly, C(2)-ceramide inhibited oxidase activity and degranulation of PMNs treated with cytochalasin B followed by FMLP, and DiC10 restored function. In contrast, C(2)-ceramide did not inhibit phosphorylation of p47phox or p38 mitogen-activated protein kinase, or translocation of p47phox, PLD-containing organelles, adenosine diphosphate-ribosylation factor 1, RhoA, protein kinase C (PKC)-beta or PKC-alpha to the plasma membrane in G-CSF or cytochalasin B-treated, FMLP-activated PMNs. PLD activity increased by 3-fold in G-CSF-primed PMNs stimulated by FMLP and by 30-fold in cytochalasin B-treated PMNs stimulated by FMLP. Both PLD activities were completely inhibited by 10 microM C(2)-ceramide. In conclusion, superoxide, gelatinase, and lactoferrin release require activation of the PLD pathway in primed PMNs and cytochalasin B-treated PMNs. Ceramide may affect protein interactions with PLD in the plasma membrane, thereby attenuating PMN activation.     

Sequential administration of recombinant human interleukin-3 and granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for malignant lymphoma: a phase I/II multicenter study

Preclinical studies of recombinant human interleukin-3 (rhIL-3) and granulocyte-macrophage colony-stimulating factor (rhGM-CSF) have shown enhancement of multilineage hematopoiesis when administered sequentially. This study was designed to evaluate the safety, tolerability, and biologic effects of sequential administration of rhIL- 3 and rhGM-CSF after marrow ablative cytotoxic therapy and autologous bone marrow transplantation (ABMT) for patients with malignant lymphoma. Thirty-seven patients (20 patients with non-Hodgkin's lymphoma and 17 patients with Hodgkin's disease) received one of four different treatment regimens before ABMT. Patients were entered in one of four study groups to receive rhIL-3 (2.5 or 5.0 micrograms/kg/day) administered by subcutaneous injection for either 5 or 10 days starting 4 hours after the marrow infusion. Twenty-four hours after the last dose of rhIL-3, rhGM-CSF (250 micrograms/m2/d as a 2-hour intravenous infusion) administration was initiated. rhGM-CSF was administered daily until the absolute neutrophil count (ANC) was > or = 1,500/microL for 3 consecutive days or until day 27 posttransplant. The most frequent adverse events in the trial included nausea, fever, diarrhea, mucositis, vomiting, rash, edema, chills, abdominal pain, and tachycardia. Three patients were removed from the study because of chest, skeletal, and abdominal pain felt to be probably related to study drug. Four patients died during the study period because of complications unrelated to either rhIL-3 or rhGM-CSF. The median time to recovery of neutrophils (ANC > or = 500/microL) and platelets (platelet count > or = 20,000/microL) was 14 and 15 days, respectively. There were fewer days of platelet transfusions than seen in historical control groups using rhGM-CSF, rhG-CSF, or rhIL-3 alone. In addition, there were fewer days of red blood cell transfusions compared with historical controls using no cytokines or rhGM-CSF. These data indicate that the sequential administration of rhIL-3 and rhGM-CSF after ABMT is safe and generally well-tolerated and results in rapid recovery of multilineage hematopoiesis.     

Regulation of human eosinophil precursor production by cytokines: a comparison of recombinant human interleukin-1 (rhIL-1), rhIL-3, rhIL-5, rhIL-6, and rh granulocyte-macrophage colony-stimulating factor

The effect of a panel of recombinant human (rh) cytokines on the generation of human eosinophil precursors was assessed using a two-step culture technique. Normal human bone marrow was preincubated with different cytokine combinations in liquid culture before assessment of the number of eosinophil progenitors, which give rise to eosinophil colony-forming units (CFU-Eo) on secondary semi-solid culture with either interleukin-5 (IL-5), IL-3, or granulocyte-macrophage colony-stimulating factor. rhIL-3 or rhGM-CSF, but not rhIL-5, increased the number of CFU-Eo. CFU-Eo production by rhIL-3 or rhGMCSF was maximal after 7 days' preincubation. Neither rhIL-1 or rhIL-6 acted on eosinophil precursors, either alone or in combination with rhIL-5, rhIL-3, or rhGM-CSF. A similar spectrum of activity of the cytokines was demonstrated whether rhIL-5, rhIL-3, or rhGM-CSF was used in the secondary cultures as the eosinophil CSF. However, rhIL-3 induced relatively more rhIL-5-responsive CFU-Eo than rhIL-3-responsive CFU-Eo, suggesting that rhIL-3 is pushing progenitors into an rhIL-5-responsive compartment.     

Tumor necrosis factor primes neutrophils by shortening the lag period of the respiratory burst

Pretreatment of neutrophils (PMNs) with low-dose tumor necrosis factor (TNF) enhances their capacity to produce oxidant radicals after stimulation with a variety of agents ('priming'). We used a continuous cytochrome C assay to investigate the superoxide production by human PMNs primed with TNF and subsequently stimulated with phorbol myristate acetate (PMA). There was no difference in the maximum rate of superoxide production by primed and unprimed PMNs stimulated with either high (2 x 10(-6) M) or low levels of PMA (2 x 10(-8) M). Following stimulation with high levels of PMA, primed PMNs demonstrated a significantly shorter lag period than unprimed cells (103.3 +/- 14.4 vs. 142.1 +/- 21.7 seconds) and larger amounts of superoxide generated in the intervals between 100 seconds (3.1 +/- 0.5 vs. 1.7 +/- 0.3 nmol/10(6) PMNs) and 300 seconds (14.9 +/- 1.2 vs. 12.2 +/- 1.1). Primed cells stimulated with low levels of PMA (2 x 10(-8) M) displayed a significantly shorter lag period (1225.8 +/- 96.8 vs. 1573.8 +/- 74.3 seconds) and a greater production of superoxide between 1300 seconds (5.4 +/- 0.9 vs. 3.0 +/- 0.4 nmol/10(6) PMNs) and 1900 seconds (25.7 +/- 4.3 vs. 14.9 +/- 2.4) than unprimed cells. These results indicate that priming of PMNs with TNF increases superoxide production during the early phase of the respiratory burst through a shortening of the post-stimulation lag period.     

Interleukin-4 inhibits human macrophage activation by tumor necrosis factor, granulocyte-monocyte colony-stimulating factor, and interleukin-3 for antileishmanial activity and oxidative burst capacity.

Interleukin (IL)-4 has been implicated in the pathogenesis of leishmaniasis in a murine model. Experiments were done to examine the effect of IL-4 on cytokine activation of macrophages. Interferon (IFN)-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF alpha), and IL-3 activate macrophages to inhibit replication of leishmaniae. IL-4 abrogated in a dose- and time-dependent manner the induction of antileishmanial activity by these cytokines. The depression of oxidative burst capacity is one mechanism by which IL-4 inhibits macrophage activation. IL-4 diminished in a dose- and time-dependent manner the TNF alpha enhancement of oxidative capacity. Pretreatment with IL-4 for 48, 24, or 0 h, respectively, inhibited the generation of superoxide induced by TNF alpha by 90%, 60%, and 40%. Furthermore, IL-4 abrogated the enhancement of oxidative capacity by IFN-gamma, GM-CSF, and IL-3. These data suggest that IL-4 is a potent deactivator of macrophage antimicrobial functions and may contribute to the pathogenesis of visceral leishmaniasis.     

Recombinant human stem cell factor synergises with GM-CSF, G-CSF, IL-3 and epo to stimulate human progenitor cells of the myeloid and erythroid lineages.

The cDNA for human stem cell factor (hSCF) has been cloned and expressed in mammalian and bacterial hosts and recombinant protein purified. We have examined the stimulatory effect of recombinant human SCF (rhSCF) on human bone marrow cells alone and in combination with recombinant human colony stimulating factors (CSFs) and erythropoietin (rhEpo). RhSCF alone resulted in no significant colony formation, however, in the presence of rhGM-CSF, rhG-CSF or rhIL-3, rhSCF stimulated a synergistic increase in colony numbers. In addition, increased colony size was stimulated by all combinations. The morphology of cells in the colonies obtained with the CSFs plus rhSCF was identical to the morphology obtained with rhGM-CSF, rhG-CSF or rhIL-3 alone. RhEpo also synergised with rhSCF to stimulate the formation of large compact hemoglobinized colonies which stained positive for spectrin and transferrin receptor and had a morphological appearance consistent with normoblasts. RhSCF stimulation of low density non-adherent, antibody depleted, CD34+ cells suggests that rhSCF directly stimulates progenitor cells capable of myeloid and erythroid differentiation.     

Inhibitory effect of granulocyte-macrophage colony-stimulating factor therapy on the generation of natural killer cells.

We investigated the effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) therapy on the natural killer (NK) cell lineage in patients with aplastic anemia and myelodysplastic syndrome. Selected bone marrow (BM) cells were prepared by the elimination of nylon wool-adherent cells and mature T and NK cells from BM cells. The frequency of BM NK progenitors relative to BM cells selected was significantly decreased 4 weeks after the start of rhGM-CSF therapy (P less than .01), while the peripheral blood NK cell count and NK activity were also significantly decreased (P less than .05). A return to the pretreatment levels was seen 4 weeks after the cessation of treatment in all cases. No suppressive effect was noted in the patients who received rhG-CSF therapy. These results suggest that rhGM-CSF therapy suppresses the generation of NK cells from human BM NK progenitors.     

Lack of effect of granulocyte-macrophage and granulocyte colony-stimulating factors on cultured human endothelial cells.

The hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), enhance the effector functions of mature myeloid cells, including the interaction with vascular endothelium. We examined the direct effect of recombinant human GM-CSF (rhGM-CSF) and recombinant human G-CSF (rhG-CSF) on the growth and function of cultured human umbilical vein endothelial cells (HUVEC). Endothelial cell growth supplement (ECGS) increased the proliferation of passaged and primary cells by 305% +/- 45% (mean +/- SEM, n = 5, P less than .01) over control cells at 4 days; GM-CSF and G-CSF had no effect. Endothelial cell procoagulant activity was increased after 4-hour incubation with recombinant interleukin-1 beta (IL-1 beta) 10 U/mL and recombinant tumor necrosis factor (TNF) 10 U/mL to 1,721% +/- 376% (n = 7, P less than .005) and 247% +/- 71% (n = 4) of control levels, respectively. gamma-Interferon (gamma-IFN) 50 U/mL had no direct effect of its own but was able to prime the response to IL-1 beta. There was no direct or priming effect of GM-CSF (1 ng to 1 microgram/mL) on the expression of procoagulant activity in endothelial cells. GM-CSF and G-CSF (1 ng/mL to 1 microgram/mL) had no effect on the expression of either tissue plasminogen activator (tPA) or plasminogen activator inhibitor-1 (PAI-1) by endothelial cells. The secretion of tPA by endothelial cells was increased, however, after 24-hour incubation with thrombin 4 U/mL (314% +/- 72% of control levels, n = 5, P less than .025). The production of PAI-1 was increased by TNF 200 U/mL (241% +/- 44% of control, n = 3, P less than .005), thrombin 4 U/mL (180% +/- 12% of control, n = 5, P less than .0005) and IL-1 beta 10 U/mL (275% +/- 44% of controls, n = 5, P less than .0005). In four experiments, endothelial cells showed no specific binding of 125I-GM-CSF, whereas peripheral blood (PB) neutrophils demonstrated the presence of 802 +/- 78 high-affinity receptors for GM-CSF. Thus, we found no effect of rhGM-CSF or rhG-CSF on the proliferation activities by these cells. These findings are in accordance with the lack of demonstrable receptors for GM-CSF on cultured HUVEC.     

Sequential in vivo treatment with two recombinant human hematopoietic growth factors (interleukin-3 and granulocyte-macrophage colony-stimulating factor) as a new therapeutic modality to stimulate hematopoiesis: results of a phase I study.

In a phase I study, the sequentially administered combination of recombinant human interleukin-3 (rhIL-3) and rhGM-CSF was compared with treatment with rhIL-3 alone in 15 patients with advanced tumors but normal hematopoiesis. Patients were initially treated with rhIL-3 for 15 days. After a treatment-free interval, the patients received a second 5-day cycle of rhIL-3 at an identical dosage, immediately followed by a 10-day course of rhGM-CSF, to assess the toxicity and biologic effects of this sequential rhIL-3/rhGM-CSF combination. rhIL-3 doses tested were 125, and 250 micrograms/m2, whereas rhGM-CSF was administered at a daily dosage of 250 micrograms/m2. Both cytokines were administered by subcutaneous (SC) bolus injection. rhIL-3/rhGM-CSF treatment was more effective than rhIL-3 but equally effective to each other in increasing peripheral leukocyte counts, especially neutrophilic and eosinophilic granulocyte counts. In contrast, both modes of cytokine therapy raised the platelet counts to the same degree. rhIL-3/GM-CSF treatment was more effective than rhIL-3 in increasing the number of circulating hematopoietic progenitor cells BFU-E and CFU-GM. High-dose rhIL-3, but not low-dose rhIL-3, was as effective as the rhIL-3/rhGM-CSF combinations in increasing the number of circulating CFU-GEMM. The increase in absolute neutrophil counts correlated with the increase in the number of circulating CFU-GM. Side effects, mainly fever, headache, flushing, and sweating, were generally mild, but in two patients the occurrence of chills, rigor, and dyspnea after initiation of GM-CSF treatment necessitated dose reduction and discontinuation, respectively. These results indicate that sequential treatment with rhIL-3 and rhGM-CSF is as effective as single-factor treatment with rhIL-3 in stimulating platelet counts, whereas the effect of combination therapy on neutrophil counts and circulating progenitor cells is superior.