Granulocyte—macrophage colony stimulating factor in acute non-lymphocytic leukaemia

Abstract. Background. Granulocyte-macrophage colony stimulating factor (GM-CSF) is required to maintain and to regulate granulocyte and monocyte productions. It is potentially useful for accelerating and enhancing haemopoietic recovery and neutrophil function. Results. In acute non-lymphocytic leukaemia (ANLL) the dependence of blast cells on GM-CSF and its role in the development and maintenance of leukaemia are still poorly defined. In vitro exposure of fresh leukaemic blast cells to a wide range of concentrations of GM-CSF leads to a stimulation of cell proliferation in the majority of cases but does not induce any detectable maturation. Conclusions. Granulocyte-macrophage colony stimulating factor may be used in the management of ANLL and the myelodysplastic syndrome (MDS) with the aim of: (i) accelerating the recovery of normal haemopoiesis; (ii) increasing cell killing by chemotherapy; and (iii) inducing cell maturation. Therefore the application of GM-CSF can potentially improve treatment outcome in ANLL and is worth testing in a clinical setting.     

Intensive chemotherapy for adult acute lymphoblastic leukaemia given with or without granulocyte-macrophage colony stimulating factor

 Twenty-six patients with newly diagnosed ALL (age range 15–49 years, median 32 years) received treatment comprising: cycles 1 and 2: adriamycin 30 mg/m² days 1–3, vincristine: 2 mg days 1, 8, and 15, with prednisolone 40 mg daily, given until complete remission (CR). l-asparaginase 10000 units/m², days 1–14, was given only with the first cycle. Cycle 3 consisted of 100 mg/m² etoposide orally, days 1–5, and 1 gm/m² bd cytosine arabinoside (ara-C) days 1–5. Cycles 1–3 were then repeated. Intrathecal methotrexate (MTX) 12.5 mg was given on day 1 of each treatment cycle. The first 12 consecutive patients received this chemotherapy alone, the subsequent 14 received, in addition, 3 μg/kg GM-CSF subcutaneously, from day 4 of cycles 1, 2, 4, and 5 (and from day 6 of cycles 3 and 6) until the absolute neutrophil count had reached 0.5×10⁹/l. All patients in whom CR was achieved then received prophylactic cranial irradiation. With the exception of those with T-ALL, this was followed by oral maintenance therapy consisting of 6-mercaptopurine, MTX, and cyclophosphamide for 3 years. Patients receiving GM-CSF did not have shorter intercycle times or a lower incidence of documented infections than those who did not receive it. The CR rate was 89% overall - uninfluenced by GM-CSF, but higher than that achieved previously at St Bartholomew's Hospital in an equivalent age-group. Received: 15 April 1996 / Accepted: 2 September 1996     

Haematologic effects of granulocyte-macrophage colony stimulating factor in a patient with thiamazole-induced agranulocytosis.

A 61-year-old female patient, treated for hyperthyroidism with thiamazole, developed a severe maturation arrest in the granulocytic lineage and a total agranulocytosis. Subcutaneous GM-CSF was started immediately and given for 6 days. Bone marrow samples were taken before GM-CSF therapy and on days 3 and 8. An increased number of colonies per 10(5) bone marrow cells documented before institution of GM-CSF treatment was followed by a gradual decline to normal values. An increase of granulocyte count to > 0.5 x 10(9) l-1 was recorded on the 4th day of treatment and was preceded by an increase in the number of immature granulocyte precursors in the bone marrow on day 3. The patient was discharged on day 8 and experienced no adverse effects of GM-CSF treatment. Haematopoietic growth factor therapy has a place in the management of patients with drug induced neutropenia/agranulocytosis, which should be further delineated by prospective studies.     

Sources and nature of granulocyte-macrophage colony stimulating factor in fetal mice.

At the earliest stages of fetal hepatic hemopoiesis in CBA mice (11-12 days gestation), colony stimulating activity could be found only in peripheral blood, yolk-sac fluid and media conditioned by yolk-sacs (YSCM). The colony stimulating factor (GM-CSF) from YSCM was able to be concentrated by absorption to DEAE-cellulose and subsequent elution. Titration of this material produced a sigmoid dose-response curve in agar cultures of adult CBA bone marrow cells. Unlike the high proportion of granulocyte colonies stimulated by the GM-CSF from mouse lung conditioned medium, all concentrations of YSCM produced a high proportion of macrophage colonies after 7 days of incubation. Mixing experiments eliminated the possibility that a specific inhibitor preventing granulocyte differentiation was present in YSCM. Fetal liver cells were relatively unresponsive to YSCM, but their ability to respond increased with gestational age. When stimulated by YSCM, fetal liver colony forming cells from mice of all gestational ages produced more than 90% macrophage colonies after 7 days of incubation. The experimental data suggest that the proliferation and differentiation of granulocyte and macrophage precursors in the early fetal liver could be controlled by a fetal type of GM-CSF favoring macrophage production.     

Sulfasalazine induced agranulocytosis treated with granulocyte-macrophage colony stimulating factor.

We report the use of granulocyte-macrophage colony stimulating factor (GM-CSF) in a case of rheumatoid arthritis with sulfasalazine induced agranulocytosis, leading to a rapid bone marrow recovery within 7 days. This case and 2 others reported in the literature emphasize the need for further research into the possible role of GM-CSF in the treatment of drug induced agranulocytosis.     

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.     

Thymomodulin increases release of granulocyte-macrophage colony stimulating factor and of tumour necrosis factor in vitro.

To evaluate the effects of thymomodulin (TMD), a thymic biological response modifier derived from calf thymus, on the release of various cytokines involved in the lung immune reactions, human alveolar macrophages (AM) and peripheral blood lymphocytes (PBL) were cultured either alone or in co-cultures. In co-cultures of AM with PBL, TMD did not induce any change in gamma-interferon (gamma-IFN) and interleukin-1 (IL-1) secretion, while it was able to increase the level, of tumour necrosis factor (TNF) (TMD 100 micrograms.m-1, p < 0.05 vs control cultures), and of granulocyte macrophage colony-stimulating factor (GM-CSF) (TMD 1, 10 and 100 micrograms.ml-1 p < 0.05, < 0.05 and < 0.01, respectively, vs control cultures). In cultures of AM alone, TMD did not induce changes in the levels of any of the tested cytokines, whilst in supernatants from PBL lymphocyte cultures, TMD at 1, 10 and 100 micrograms.ml-1 increased the amounts of GM-CSF (p < 0.01 each comparison vs control cultures). Thus, TMD seems to directly stimulate lymphocytes to secrete GM-CSF and to modulate macrophage-lymphocyte interactions, resulting in the release of TNF and GM-CSF.     

Purification and properties of bacterially synthesized human granulocyte-macrophage colony stimulating factor

Human granulocyte-macrophage colony stimulating factor (GM-CSF) has been synthesized in high yield using a temperature inducible plasmid in Escherichia coli. The human GM-CSF is readily isolated from the bacterial proteins because of its differential solubility and chromatographic properties. The bacterially synthesized form of the human GM-CSF contains an extra methionine residue at position 1, but otherwise it is identical to the polypeptide predicted from the cDNA sequence. The specific activity of 2.9 X 10(7) units/mg of protein for purified bacterially synthesized human GM-CSF indicates that despite the lack of glycosylation, the molecule is substantially in its native conformation. This molecule stimulated the same number and type of both seven- and 14-day human bone marrow colonies as the CSF alpha preparation from human placental conditioned medium. Human GM-CSF had no activity on murine bone marrow or murine leukemic cells. There was no detectable, direct stimulation of adult human erythroid burst forming units (BFU-E) by the bacterially synthesized human GM-CSF. Although impure preparations containing native human GM-CSF (eg, human placental conditioned medium) stimulated the formation of mixed colonies, even in the presence of erythropoietin, the bacterially synthesized human GM-CSF failed to stimulate the formation of mixed colonies from adult human bone marrow cells. The bacterially synthesized human GM-CSF increased N-formyl-methionyl-leucyl- phenylalanine (FMLP)-induced superoxide production and lysozyme secretion. Antibody-dependent cytotoxicity and phagocytosis by human neutrophils was stimulated by the bacterially synthesized human GM-CSF and eosinophils were also activated in the antibody-dependent cytotoxicity assay.     

Treatment with granulocyte-macrophage colony stimulating factor and the adult respiratory distress syndrome.

We report a case of a patient who developed a fatal adult respiratory distress syndrome (ARDS) during treatment with rh granulocyte-macrophage colony stimulating factor (rhGM-CSF) (250 mcg/m2/day s.c.) and low-dose cytosine-arabinoside (Ara-C) (20 mg/m2/day s.c.). Several mechanisms which might explain the lung tissue damage in this patient were explored. GM-CSF increased the expression of the glycoproteins CD11B and CD18 on the surface of his neutrophils, which may have increased the adhesiveness of neutrophils to the pulmonary endothelium. In addition, GM-CSF primed the neutrophils of the patient to an enhanced release of superoxide anions. Both findings may at least partially explain why GM-CSF exerted a deleterious action on the pulmonary endothelial integrity in this patient. Other factors, such as increased platelet-activating factor production by the neutrophils or tumor necrosis factor-mediated mechanisms, may also have played a role. ARDS as a complication of low-dose Ara-C seems less plausible.     

Identification of the soluble granulocyte-macrophage colony stimulating factor receptor protein in vivo

On the basis of the finding of alternatively spliced mRNAs, the alpha-subunit of the receptor for GM-CSF is thought to exist in both a membrane spanning (tmGMRalpha) and a soluble form (solGMRalpha). However, only limited data has been available to support that the solGMRalpha protein product exists in vivo. We hypothesized that hematopoietic cells bearing tmGMRalpha would have the potential to also produce solGMRalpha. To test this hypothesis we examined media conditioned by candidate cells using functional, biochemical, and immunologic means. Three human leukemic cell lines that express tmGMRalpha (HL60, U937, THP1) were shown to secrete GM-CSF binding activity and a solGMRalpha-specific band by Western blot, whereas a tmGMRalpha-negative cell line (K562) did not. By the same analyses, leukapheresis products collected for autologous and allogeneic stem cell transplants and media conditioned by freshly isolated human neutrophils also contained solGMRalpha. The solGMRalpha protein in vivo displayed the same dissociation constant (Kd = 2-5 nmol) as that of recombinant solGMRalpha. A human solGMRalpha ELISA was developed that confirmed the presence of solGMRalpha in supernatant conditioned by the tmGMRalpha-positive leukemic cell lines, hematopoietic progenitor cells, and neutrophils. Furthermore, the ELISA demonstrated a steady state level of solGMRalpha in normal human plasma (36 +/- 17 pmol) and provided data suggesting that plasma solGMRalpha levels can be elevated in acute myeloid leukemias. (Blood. 2000;95:461-469)     

Sweet's syndrome associated with sargramostim (granulocyte-macrophage colony stimulating factor) treatment

Sweet's syndrome is an acute febrile neutrophilic dermatosis that is a known complication of the administration of filgrastim, a drug that causes increased neutrophil proliferation and differentiation. This complication has not previously been reported during treatment with sargramostim, a hematopoietic cytokine with activity that overlaps filgrastim. We report a case of Sweet's syndrome in association with sargramostim treatment following chemotherapy for acute myelogenous leukemia. A suspected second episode occurred after subsequent chemotherapy and sargramostim treatment. Physicians should be aware of this possible association because the signs and symptoms of Sweet's syndrome are easily mistaken as being due to infection.     

Recombinant granulocyte-macrophage colony stimulating factor followed by immunosuppressive therapy for aplastic anaemia

Summary. Seventeen patients with aplastic anaemia were treated with recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) for 14 d. Nonrespond-ing patients were then treated with anti-human thymocyte globulin (ATG), methylprednisolone and oxymetholone. Side-effects of rhGM-CSF included fever, nausea and vomiting, diarrhoea, bone pain, headache and chills. Two patients had sustained trilineage haemopoietic recovery after receiving only rhGM-CSF. Of 11 patients who received immunosuppressive therapy, there was one complete response, two partial responses, one minimal response, and seven nonres-ponses. Actuarial survival at 2 years is 64%. Early administration of rhGM-CSF had no apparent effect on subsequent response to immunosuppressive therapy.     

Bone marrow response to chemotherapy in acute lymphocytic leukaemia and acute non-lymphocytic leukaemia

Histopathologic changes in core bone marrow biopsies were reviewed in 33 patients with acute leukaemia during chemotherapy to compare the changes in acute lymphocytic leukaemia (ALL) with acute non-lymphocytic leukaemia (ANLL). Cellular, stromal, and bony changes were evaluated with regard to diagnosis and time of biopsy from initiation of chemotherapy. A significant difference was noted in the plasma cell response. Plasmacytosis was present in 19/19 cases of ANLL, but in only 2/14 cases of ALL. Cellular depletion was also significantly less frequent in ALL. Other stromal changes such as haemorrhage, dilatation of sinusoids and fat regeneration, as well as osteoblastic bone activity occurred with similar frequencies in all cases of treated acute leukaemia. Fibrosis, necrosis, and serous atrophy were uncommon. Differing chemotherapeutic regimens and differing patient ages were both correlated with the plasma cell response, but not with the difference in cellular depletion.     

Healing of chronic leg ulcers in diabetic necrobiosis lipoidica with local granulocyte-macrophage colony stimulating factor treatment.

Two young insulin-dependent diabetic patients suffering from chronic nonhealing leg ulcers of necrobiosis lipoidica diabeticorum were treated by applying topically recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) on the ulcer repetitively during 10 weeks. Evaluation of ulcer size was assessed with clinical examinations at 1-week or 2-week intervals. Topical GM-CSF healed the ulcers of both patients in 10 weeks. Decrease in the size of the ulcers was already evident after the first topical applications. During follow-up, the ulcers have remained healed for more than 3 years. This excellent treatment result suggests that topically applied GM-CSF may be a valuable drug for chronic, nonhealing ulcers in patients with diabetes.     

Enhanced expression of the granulocyte-macrophage colony stimulating factor gene in acute myelocytic leukemia cells following in vitro blast cell enrichment

Expression of the granulocyte-macrophage colony-stimulating factor (GM- CSF) gene in acute myelocytic leukemia (AML) was assayed by Northern blot analysis. GM-CSF messenger RNA (mRNA) was detected in the freshly obtained mononuclear cells of only one of 48 cases of AML, in contrast with recent reports that GM-CSF mRNA might be detected in half of the cases of AML when RNA is prepared from T-cell- and monocyte-depleted leukemic cells. We did find, however, that expression of the GM-CSF gene was detectable in five of ten cases after in vitro T-cell and monocyte depletion steps. Additional studies suggest that expression of GM-CSF in the bone marrow of the one positive case, rather than being autonomous, was under exogenous control, possibly by a paracrine factor secreted by marrow stromal cells. These studies emphasize the potential for altering in vivo patterns of gene expression by in vitro cell manipulation.     

The in vitro effects of stem cell factor, interleukin 3 and granulocyte-macrophage colony stimulating factor on haemopoietic progenitor cells from premature infants

Summary. Circulating haemopoietic progenitor cells from premature infants were assessed for their ability to respond to interleukin 3, granulocyte-macrophage colony stimulating factor and stem cell factor (SCF) in vitro. All three cytokines increased the number of colonies derived from burst forming units erythroid (BFU-E), colony forming units granulocyte-macrophage (CFU-GM) and multi-lineage progenitors (CFU-Mix) grown in the presence of erythropoietin (Epo). The size and haemoglobin content of BFU-E derived colonies also increased in the presence of the cytokines. Of those tested, SCF was found to be the most potent additive to Epo for the enhanced growth of BFU-E and CFU-Mix. In short-term liquid cultures without Epo, SCF alone induced globin synthesizing cells. Progenitors from premature infants were at least as responsive to all three cytokines as those from healthy adults. The use of SCF in combination with Epo in the prevention or treatment of anaemia in premature infants warrants further investigation.     

Polyethylene glycol (PEG) modification of granulocyte-macrophage colony stimulating factor (GM-CSF) enhances neutrophil priming activity but not colony stimulating activity

PEG-modified proteins have numerous advantages over their unmodified counterparts (increased half life, reduced antigenicity, improved solubility), but almost without exception, they show a modest to marked reduction in biological or enzymatic activity. However, while investigating a new protocol for the preparation of PEG-proteins, we compared PEG-modified and unmodified GM-CSF with respect to their polymorphonuclear neutrophil granulocyte (PMN) priming activities. PEG-GM-CSF was unexpectedly more active than GM-CSF in its ability to prime neutrophils to respond to the synthetic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP) with an oxidative burst (assessed both by nitroblue tetrazolium reduction and ferricytochrome c reduction). These results were in contrast to the findings for colony stimulating activity and with GM-CSF induced thymidine uptake, where the biological activity was unchanged or reduced. The enhanced neutrophil priming activity of PEG-GM-CSF was confirmed using FPLC fractionated PEG-modified GM-CSF. This showed changes in the bioactivity profile consistent with both the shift in protein elution profile and enhanced activity of the PEG-modified material (reflected in the increased area under the bioactivity curve). We also excluded a neutrophil priming action for PEG-modified fetal calf serum proteins, carrier proteins and 'irrelevant' cytokine, erythropoietin. The dissociation of the two bioactivities was confirmed using individual FPLC fractions. These results suggest the presence of differences in either binding, receptor/ligand processing or signal transduction for neutrophils versus progenitors, that are differentially affected by PEG-modification of GM-CSF. The demonstration that PEG-modification can partially dissociate two biological activities suggests the feasibility of using PEG-modification to produce proteins with subtly altered spectra of biological activity and hence new ranges of clinical applications.     

Autoantibodies against granulocyte-macrophage colony stimulating factor and interleukin-3 are rare in patients with Felty's syndrome

OBJECTIVES: Antibodies against granulocyte colony stimulating factor are frequently found in patients with Felty's syndrome (FS). In this study, we examined the prevalence of antibodies against two other granulopoietic cytokines: granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL3). METHODS: Sera of 32 patients with FS, 20 normocytic patients with rheumatoid arthritis (RA), and 72 healthy individuals were screened for the presence of antibodies against GM-CSF and IL3 by ELISA and bioassays, using the human erythroleukaemia cell line TF-1. RESULTS: In two of the 32 patients with FS, antibodies to GM-CSF and IL3 were detectable by ELISA. Binding anti-GM-CSF antibodies were also detected in one of the 72 healthy controls, while in another healthy subject and in one of the patients with normocytic RA, anti-IL3 antibodies were present. Serum from one of the two patients with FS who tested positive for anti-IL3 and anti-GM-CSF antibodies by ELISA showed strong neutralising capacity to the biological effect of IL3, but not to GM-CSF in vitro. Patients with FS had significantly higher serum levels of GM-CSF (median; 2.82 pg/mL; interquartile range 2.64-3.19 pg/mL) compared with patients with RA (2.52 pg/mL; 2.28-2.72 pg/mL; p = 0.012) and healthy controls (2.23 pg/mL; 2.04-2.52; p<0.001). In addition, serum levels of IL3 in patients were significantly higher in FS (10.05 pg/mL; 8.94-11.98) compared with controls (4.79 pg/mL; 3.72-7.22; p<0.001), but not compared with RA patients (9.52 pg/mL; 8.32-10.42; p = 0.17). CONCLUSIONS: Antibodies to GM-CSF and IL3 are rare in patients with FS and RA and in healthy subjects. In individual patients with FS, the presence of neutralising anti-IL3 antibodies may contribute to the development of cytopenia.