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.     

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.     

Recombinant human interleukin-3 and recombinant human granulocyte-macrophage colony-stimulating factor administered in vivo after high-dose cyclophosphamide cancer chemotherapy: effect on hematopoiesis and microenvironment in human bone marrow.

The effects on bone marrow (BM) cell proliferation and differentiation of recombinant human interleukin-3 (rhIL-3) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) administered after high-dose (7 g/m2/d) cyclophosphamide (HD-CTX) chemotherapy were studied in nine patients with malignancies without BM involvement and in three control patients. rhIL-3 at a dose of 1 to 5 micrograms/kg/day was administered for 14 to 18 days by continuous intravenous (i.v.) infusion and rhGM-CSF was administered at a dose of 5.5 micrograms/kg/day for 14 days. Changes induced by cytokine treatment were assessed by morphoimmunohistochemical analysis of BM biopsies. Comparison was made in the cytokine-treated groups and with control patients who received HD-CTX alone. BM cellularity and the myeloid/erythroid (ME) ratio were lower in rhIL-3-treated than in rhGM-CSF-treated patients, but in both groups it was significantly higher than in the controls. The proportion of BM cells stained by PC10, a monoclonal antibody (MoAb) recognizing a proliferation-associated nuclear protein (PCNA), increased from 6.78% to 21.18% (P less than .02) after rhIL-3, and from 5% to 35.33% (P less than .001) after rhGM-CSF; no increase was observed in the control group. The frequency of CD34+ BM cells was unchanged after rhIL-3 (P = NS) and decreased after rhGM-CSF (P less than .001). In both groups, most of the PC10+ cells were represented by promyelocytes and myelocytes with no increase in blast cell numbers. rhIL-3-treated BM showed an increased number of megakaryocytes and increased proliferative activity of erythroid cells as compared with rhGM-CSF cases. BM stroma changes observed in both treated groups included endothelial cell proliferation, increased BM macrophage concentration, and increase in BM fibroblasts as detected with an anti-nerve growth factor receptor antibody. In most rhIL-3-treated cases, BM fibrosis developed after treatment. The same effect was not observed in rhGM-CSF patients.     

Decreased frequency of bone marrow NK progenitors in aplastic anaemia

Twelve patients with aplastic anaemia were studied with regard to the frequency of NK progenitors in the bone marrow (BM) to investigate the mechanism of depressed NK activity and low NK cell count in the peripheral blood. NK cell (CD16+ cell) count and NK (K562) activity were significantly decreased (P less than 0.02 and P less than 0.001, respectively) in the patients as compared to eight healthy control subjects. Nylon wool non-adherent (NW-NA) BM mononuclear cells (MNC) of each patient and control were prepared. Mature T and NK cells were extensively depleted by sheep red cell rosette formation followed by a centrifugation on Ficoll-Hypaque and monoclonal antibody mediated complement dependent cytolysis. Those selected BM cells were cultured in the presence of recombinant interleukin 2 (rIL2). Generation of NK activity was significantly decreased (P less than 0.01) in the patients with aplastic anaemia. Frequency of NK progenitors in the selected BM cells assayed by the limiting dilution method was significantly decreased in those patients (P less than 0.05). The frequency of BM NK progenitors relative to NW-NA BM cells were related to NK cell count (P less than 0.01). Those results indicate that depressed NK activity in aplastic anaemia is closely related to decreased NK cell count which is probably due to decreased production of NK cells in the BM.     

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.     

Kinetics of hematopoiesis in bone marrow cultures from patients with chronic myeloid leukemia: effect of recombinant cytokines in dexter-type long-term cultures

Chronic myeloid leukemia (CML) is a hematological neoplasia that results from the transformation of a hematopoietic stem cell. It is characterized by the expansion of the myeloid lineage, which results in the accumulation of mature and immature granulocytes in peripheral blood and bone marrow. However, when CML marrow cells are cultured in Dexter-type long-term cultures (LTMC) hematopoiesis is defective and can be sustained for only a few weeks. One possible explanation for the deficient growth of hematopoietic cells in CML LTMC is that some factors that act as key regulators of hematopoiesis are absent in this experimental system. Thus, we tested this hypothesis by adding recombinant cytokines to these cultures. As a first approach, we added recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), rhGranulocyte-CSF (rhG-CSF) and rhErythropoietin (rhEPO); each factor was added individually once a week. Addition of rhGM-CSF and rhG-CSF resulted in a significant increase in the levels of nucleated cells and myeloid progenitors; the highest effects were seen in the presence of rhGM-CSF. Interestingly, such a cytokine also induced a significant decrease in the levels of erythroid progenitors. Recombinant hEPO had no significant effects on nucleated cells or myeloid progenitors, however, it induced a significant, although transient, increase in the levels of erythroid cells. The above results indicate that the hematopoietic regulators used here (rhGM-CSF, rhG-CSF and rhEPO) are capable of stimulating the growth of hematopoietic cells in LTMC from CML patients. Thus, this study demonstrates that it is, indeed, possible to manipulate CML LTMC by the addition of recombinant cytokines; this observation may be of particular relevance, since this in vitro experimental system has already been used as a method for purging of leukemic cells in autologous transplant settings. By using specific recombinant hematopoietic modulators it might be possible to make LTMC a more efficient system for such a clinical purpose.     

Hematopoiesis

Chronic myeloid leukemia (CML) is a hematological neoplasia that results from the transformation of a hematopoietic stem cell. It is characterized by the expansion of the myeloid lineage, which results in the accumulation of mature and immature granulocytes in peripheral blood and bone marrow. However, when CML marrow cells are cultured in Dexter-type long-term cultures (LTMC) hematopoiesis is defective and can be sustained for only a few weeks. One possible explanation for the deficient growth of hematopoietic cells in CML LTMC is that some factors that act as key regulators of hematopoiesis are absent in this experimental system. Thus, we tested this hypothesis by adding recombinant cytokines to these cultures. As a first approach, we added recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), rhGranulocyte-CSF (rhG-CSF) and rhErythropoietin (rhEPO); each factor was added individually once a week. Addition of rhGM-CSF and rhG-CSF resulted in a significant increase in the levels of nucleated cells and myeloid progenitors; the highest effects were seen in the presence of rhGM-CSF. Interestingly, such a cytokine also induced a significant decrease in the levels of erythroid progenitors. Recombinant hEPO had no significant effects on nucleated cells or myeloid progenitors, however, it induced a significant, although transient, increase in the levels of erythroid cells. The above results indicate that the hematopoietic regulators used here (rhGM-CSF, rhG-CSF and rhEPO) are capable of stimulating the growth of hematopoietic cells in LTMC from CML patients. Thus, this study demonstrates that it is, indeed, possible to manipulate CML LTMC by the addition of recombinant cytokines; this observation may be of particular relevance, since this in vitro experimental system has already been used as a method for purging of leukemic cells in autologous transplant settings. By using specific recombinant hematopoietic modulators it might be possible to make LTMC a more efficient system for such a clinical purpose.     

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.     

Effect of recombinant human granulocyte-macrophage colony-stimulating factor administration on the lymphocyte subsets of patients with refractory aplastic anemia

Human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was administered to 14 patients with refractory aplastic anemia (AA). The effect of rhGM-CSF therapy on the lymphocyte phenotype; on the proliferative responses to the mitogen phytohemagglutinin, Candida albicans, and tetanus toxoid antigens; and on the natural killer (NK) activity of the circulating lymphocytes was studied. Samples were collected before (baseline) and twice during the rhGM-CSF administration. The absolute number of circulating lymphocytes remained relatively constant during the first period, but experienced a significant increase (P less than .001) during the second period. The increase was most prominent in the B cells (P less than .001), but the T cells (P less than .016) also increased. Detailed investigation of lymphocyte subsets showed an increase of the markers CD38 (Leu17), HLA-DR, and the transferrin receptor throughout the treatment course giving evidence of lymphoid cell activation. The NK cell activity was suppressed (P less than .008) throughout the treatment. However, proliferative responses to phytohemagglutinin, Candida antigen, and tetanus toxoid were unaffected. Although the mechanism is not yet defined, GM-CSF does induce activation and increase in absolute lymphoid cell number, especially B cells, together with a decrease in NK cytotoxicity. The implication of these immune cell changes in relation to host resistance to microorganisms remains to be established.     

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.     

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.     

Enhanced effect of mutant granulocyte-colony-stimulating factor (KW-2228) on the growth of normal and leukemic hemopoietic progenitor cells in comparison with recombinant human granulocyte-colony-stimulating factor (G-CSF).

We tested the in vitro effect of a novel granulocyte colony-stimulating factor (G-CSF) derivative (KW-2228) on the growth of G-CSF-dependent hemopoietic progenitor cells: granulocyte precursor cells (CFU-G), leukemic blast progenitors freshly obtained from 9 patients with acute myeloblastic leukemia (AML) and cells of a G-CSF-dependent human AML cell line (OCI/AML 1a). KW-2228 showed a higher stimulating effect than recombinant human G-CSF (rhCSF) on CFU-G; 3 out of 9 leukemic blast progenitors and OCI/AML 1a cells. The difference in biochemical stability between rhG-CSF and KW-2228 was considered to explain the superior colony-stimulating activity of KW-2228. The results show that KW-2228 will be a new granulopoietic factor.     

Use of recombinant human growth hormone (rhGH) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) for the mobilization and collection of CD34+ cells in poor mobilizers

The activity of recombinant human growth hormone (rhGH) in enhancing CD34(+) cell mobilization elicited by chemotherapy plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) was evaluated in 16 hard-to-mobilize patients, that is, those achieving a peak of circulating CD34+ cells 10/microL or less, or a collection of CD34(+) cells equal to or less than 2 x 10(6)/kg. Patients who had failed a first mobilization attempt with chemotherapy plus rhG-CSF (5 microg/kg/d) were remobilized with chemotherapy plus rhG-CSF and rhGH (100 microg/kg/d). As compared with rhG-CSF, the combined rhGH/rhG-CSF treatment induced significantly higher (P < or =.05) median peak values for CD34(+) cells/microL (7 versus 29), colony-forming cells (CFCs)/mL (2154 versus 28,510), and long-term culture-initiating cells (LTC-ICs)/mL (25 versus 511). Following rhG-CSF and rhGH/rhG-CSF, the median yields of CD34(+) cells per leukapheresis were 1.1 x 10(6)/kg and 2.3 x 10(6)/kg (P < or =.008), respectively; the median total collections of CD34(+) cells were 1.1 x 10(6)/kg and 6 x 10(6)/kg (P < or =.008), respectively. No specific side effect could be ascribed to rhGH, except a transient hyperglycemia occurring in 2 patients. Reinfusion of rhGH/rhG-CSF-mobilized cells following myeloablative therapy resulted in prompt hematopoietic recovery. In conclusion, our data demonstrate that in poor mobilizers addition of rhGH to rhG-CSF allows the patients to efficiently mobilize and collect CD34(+) cells with maintained functional properties.     

Use of recombinant human granulocyte-macrophage colony-stimulating factor in graft failure after bone marrow transplantation

The effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was evaluated in 37 patients with marrow graft failure after allogeneic (n = 15), autologous (n = 21), or syngeneic (n = 1) bone marrow transplantation. rhGM-CSF was administered by 2-hour infusion at doses between 60 and 1,000 micrograms/m2/d for 14 or 21 days. At doses of less than 500 micrograms/m2, rhGM-CSF was well-tolerated and did not exacerbate graft-versus-host disease in allogeneic transplant recipients. No patient with myelogenous leukemia relapsed while receiving rhGM-CSF. Twenty-one patients reached an absolute neutrophil count (ANC) greater than or equal to 0.5 x 10(9)/L within 2 weeks of starting therapy while 16 did not. None of seven patients who received chemically purged autologous marrow grafts responded to rhGM-CSF. The survival rates of GM-CSF-treated patients were significantly better than those of a historical control group.     

Treatment of idiopathic neutropenia in the elderly with recombinant human granulocyte colony-stimulating factor.

We administered recombinant human granulocyte colony-stimulating factor (rhG-CSF) intravenously for 2 weeks to 2 elderly patients with severe neutropenia. The absolute neutrophil count (ANC) recovered promptly after the initiation of rhG-CSF therapy and reached a peak (greater than 10 x 10(9)/l) on the 13th day. The ANC fell rapidly after rhG-CSF was discontinued, but it remained within the normal range after therapy. There were no side effects during the entire course of treatment. Therefore, rhG-CSF seems to be a most beneficial treatment in elderly patients with severe neutropenia.     

Peripheral blood progenitor cell mobilization in healthy donors receiving recombinant human granulocyte colony-stimulating factor

OBJECTIVE: We analyzed the incidence of primitive (LTC-IC) and committed (CFU-mix, BFU-E, CFU-GM) hematopoietic progenitors detected under steady-state conditions and upon progenitor cell mobilization in a cohort of healthy donors receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF). MATERIALS AND METHODS: Healthy donors (n = 30) of HLA-mismatched or -matched stem cell transplants were mobilized with rhG-CSF (8 microg/Kg body weight subcutaneously twice daily until completion of leukapheresis). PBPC collections were started after 4 days of rhG-CSF therapy. RESULTS: Steady-state incidence of bone marrow LTC-IC, but not committed progenitors, significantly correlated with the numbers of mobilized CD34+ cells (r = 0.6, p = 0.004), CFU-GM (r = 0.79, p = 0.0005) and CFC (r = 0.76, p = 0.001) detected after 4 days of rhG-CSF therapy. Statistically significant correlations were also found between steady-state blood CFU-GM and peak numbers of CD341 cells (r = 0.68, p = 0.001), numbers of day 4 CD341 cells (r = 0.52, p = 0.005), CFU-GM (r = 0.63, p = 0.002), and CFC (r = 0.61, p = 0.003). CONCLUSION: Our data show that in normal volunteers baseline marrow LTC-IC and blood CFU-GM correlate with rhG-CSF-mobilized PBPC. The potential clinical relevance of these findings in the identification of poor mobilizers will be tested in a prospective study.     

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) subsequent to chemotherapy improves collection of blood stem cells for autografting in patients not eligible for bone marrow harvest.

Fourteen patients with relapsed Hodgkin's disease responded to a salvage therapy with Dexa-BEAM (dexamethasone, BCNU, etoposide, Ara-C and melphalan). In seven patients a continuous i.v. infusion with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was started subsequent to Dexa-BEAM (+rhGM-CSF) while the other seven patients received no hemopoietic growth factor (-rhGM-CSF). It was our objective to study the impact of rhGM-CSF on the collection of blood-derived hemopoietic stem cells in patients with extensive prior chemo- and radiotherapy not eligible for marrow harvest. Compared to baseline, we observed a significant increase of colony-forming units granulocyte-macrophage (CFU-GM) in the peripheral blood of patients receiving rhGM-CSF (p less than 0.05). On average, the yield of total nucleated cells and CFU-GM collected per single leukapheresis was 2.2 and 2.4-fold higher in the rhGM-CSF-treated patients respectively (p less than 0.05). With rhGM-CSF the interval from the start of chemotherapy to the end of blood stem cell collection could be reduced by 6 days (p less than 0.05). Following the CBV pretransplant regimen (cyclophosphamide, BCNU, etoposide), the reinfusion of rhGM-CSF-exposed stem cells resulted in a shorter time of leukocyte recovery (p less than 0.05). The number of CFU-GM/kg body weight transplanted was found to be predictive for the time of neutrophil recovery (p less than 0.05). In patients with bone marrow hypoplasia or fibrosis, rhGM-CSF as part of an effective salvage therapy improves the collection of blood stem cells that are capable of restoring hemopoiesis after high-dose pretransplant therapy.     

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.     

Different membrane expression of CD11b and CD 14 on blood neutrophils following in vivo administration of myeloid growth factors

Summary. During the administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) or granulocyte-macrophage CSF (rhGM-CSF) we studied the early and late changes of membrane antigen density on neutrophils. RhG-CSF and rhGM-CSF both caused an early transient reduction in blood neutrophilic granulocyte-concentration within the first 30 min after treatment followed by a marked later increase during the subsequent 24 h. During the early neutropenia quantitative flow cytometry showed an associated marked increase in the density of membrane CD11b from 169 × 10³ before to 568 × 10³ A.U. per cell induced by rhGM-CSF but a non-significant change by rhG-CSF, suggesting that different mechanisms may be responsible for the transient neutropenia. The subsequent neutrophil granulocytosis was followed by a significantly (P<0.05) increased density of the CD14 antigen from 6.1 × 10³ before to 15.9 × 10³ A.U. per cell during treatment with rhG-CSF. but not by rhGM-CSF administration.
These results demonstrate that the two cytokines may affect the function of neutrophilic granulocytes in different ways. The increased expression of CD1 1b could explain some of the side-effects during treatment with rhGM-CSF. The upregulation of CD14 induced by rhG-CSF may be clinically relevant, as CD14 is an opsonic receptor for lipopolysaccharide binding proteins, acting in the defence against Gramnegative bacterial infections.     

Clinical significance of recombinant human granulocyte colony-stimulating factor (rG-CSF) in the chemotherapy of patients with malignant lymphoma]

We examined the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on neutropenia induced by chemotherapy in 10 patients with non-Hodgkin's lymphoma (NHL). The numbers of peripheral blood hematopoietic progenitors were also evaluated before and after administration of rG-CSF. Six patients received an administration of 2 micrograms/kg/body weight of rG-CSF subcutaneously for 14 days after 2nd chemotherapy. Four patients received intravenous infusion of rG-CSF (300 micrograms/body/day) for 4 days from nadir state after chemotherapy. Administration of rG-CSF from the termination of chemotherapy, markedly shortend the period of bone marrow hypoplasia induced by chemotherapy. On the other hand, administration of rhG-CSF from nadir state after chemotherapy have accelerated the recovery of neutrophil counts. In addition, this type of therapy induced 26 to 60 folds increase of peripheral blood hematopoietic progenitors. These results demonstrate the validity of administration of rhG-CSF not only in the chemotherapy of NHL, but also in peripheral blood stem cell transplantation (PBSCT).