Recombinant human granulocyte colony-stimulating factor (rhG-CSF) promotes in vitro platelet aggregation

Abstract

rhG-CSF is increasingly used for stimulation of granulopoiesis and stem cell mobilization in healthy donors for allogeneic stem cell transplantation. However, a possible association between thrombosis and rhG-CSF administration has been reported. For that reason, in this study, we investigated the effect of rhG-CSF on platelet aggregation in whole blood of 10 healthy volunteers. Three concentrations of rhG-CSF solution (1, 10 and 100 ng/ml) were prepared. Each concentration of rhG-CSF solution and a control diluent without rhG-CSF were incubated with whole blood. Incubation with rhG-CSF solutions would result in 0.1, 1.0 and 10 ng/ml rhG-CSF concentrations in the blood. After incubation, aggregation responses were evaluated with ADP (5 and 10 μM) and collagen (2 and 5 μg/ml) in whole blood. When compared to control, preincubation with all dilutions of rhG-CSF augmented aggregation of platelets induced by ADP and collagen in a statistically significant manner (p < 0.05 for all comparisons). There was also a relationship between rhG-CSF concentration (1, 10 and 100 ng/ml) and augmentation of platelet aggregation response (p < 0.0001 for 5–10 μM ADP; p < 0.0001 for 2–5 μg/ml collagen). In conclusion, this study with an in vitro model showed that rhG-CSF administration may lead to platelet hyperaggregability.     

Recombinant human granulocyte colony-stimulating factor (rhG-CSF; filgrastim) treatment of clozapine-induced agranulocytosis

Abstract. After 10 weeks of treatment with clozapine, severe agranulocytosis was diagnosed in a 33-year-old female. The patient was treated with filgrastim (granulocyte colony-stimulating factor [G-CSF]) 5 μg kg^?1 day^?1. The neutrophil count was 0.234 × 10⁹ l^?1 on admission, with a further decrease the next day to < 0.050 × 10⁹ l^?1, and this complete agranulocytosis continued for 10 days. As no response was obtained after 1 week the dosage of filgrastim was increased to 10 μg kg^?1 day^?1 with immediate improvement. A rapid and pronounced leucocytosis developed with maximal value of neutrophil granulocytes (including immature forms) of 33.108 × 10⁹ l^?1 on day 12 after admission. The patient only had minor infectious complications during the neutropenic period. In conclusion, early treatment with filgrastim seems warranted in severe cases of clozapine-induced agranulocytosis. A dosage of 10 μg kg^?1 day^?1 can be recommended.     

Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on circulating platelets

Summary The effect on platelets of rhG-CSF was studied in 20 healthy volunteers with the thrombometer, a specially developed device which is described in detail. Additionally, conventional aggregation tests were performed. Low doses of rhG-CSF enhance functional platelet activity, as shown by significant acceleration of the occlusion of the thrombometer channel. Similar results were found in conventional aggregation tests utilizing collagen for induction. At G-CSF concentrations of 0.1 and 1.0 ng/ml the time to response was significantly accelerated and the maximum response was observed in a higher proportion of platelets. However, the second phase of aggregation induced by epinephrine was significantly inhibited by 1.0 ng/ml G-CSF. The activation of platelets may be beneficial in thrombocytopenia, but it may also increase platelet turnover and platelet loss. Further investigation is needed to clarify the mechanism by which G-CSF exerts its effects on platelets.     

Clinical evaluation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in autologous bone marrow transplantation]

We administered recombinant human granulocyte colony-stimulating factor (rhG-CSF) at 5 micrograms/kg/day by intravenous drip infusion for 21 consecutive days in autologous bone marrow transplanted patients. The period of posttransplant neutropenia was markedly shortened by the rhG-CSF treatment; mean days required for neutrophil recovery (greater than 500/mm3) of 14.3 days in the rhG-CSF group (n = 21) versus 27.8 days in the historical control group (n = 11). More importantly, the numbers of febrile days between day 15 and day 28 were found to be fewer in the rG-CSF group than in control group. These effects were obtained without delay in the recovery of other blood cell series and without any side effect. We conclude that the posttransplant use of the rhG-CSF is beneficial for prevention and treatment of infectious complications after autologous bone marrow transplantation.     

Recombinant human TNF alpha stimulates production of granulocyte colony-stimulating factor

Tumor necrosis factor alpha (TNF alpha) stimulates production of granulocyte colony-stimulating factor (G-CSF) protein and mRNA in fibroblast cells. In contrast, TNF beta is at least 1,000-fold less potent than TNF alpha in increasing levels of G-CSF and GM-CSF activity and mRNA. The fibroblasts produce G-CSF and GM-CSF mRNA in a coordinate fashion after exposure to TNF. The ability of TNF alpha to stimulate production of CSF in fibroblasts may be pivotal to regulation of hematopoiesis.     

In vivo administration of granulocyte colony-stimulating factor promotes neutrophil survival in vitro

Abstract: We recently showed that recombinant human granulocyte-colony stimulating factor (rhG-CSF) maintained the viability of human neutrophils in incubation for up to 72 hours. However, it is not known whether rhG-CSF can enhance neutrophil survival in in vivo situations. To clarify this issue, we investigated neutrophil survival in vitro following in vivo injection of rhG-CSF. Neutrophils were obtained from 4 pediatric patients with malignancies and healthy adult volunteers before and after rhG-CSF administration. Neutrophils obtained before rhG-CSF treatment started to undergo apoptosis after 24 h of incubation. In contrast, the survival of neutrophils drawn after rhG-CSF administration increased by approximately 24 h. Concomitantly, the appearance of typical ladder-like DNA fragmentation was delayed. Such an increase in neutrophil survival was inhibited by coincubation with either H 7 (10 μmol/1) or H 8 (20 μmol/1), which worked as protein kinase C inhibitors. Although our study did not measure neutrophil survival in vivo directly, it provides us with further evidence that rhG-CSF may function to prolong neutrophil life expectancy in vivo.     

Acute arterial thrombosis due to platelet aggregation in a patient receiving granulocyte colony-stimulating factor

We describe a 44-year-old man with non-Hodgkin's lymphoma receiving granulocyte colony-stimulating factor (G-CSF) who developed an acute arterial thrombosis. The removed thrombus contained large amounts of platelet aggregation. A rapid increase of platelets and increased adenosine diphosphate (ADP)- and collagen-induced platelet aggregation were observed at the time of the thrombotic event. A challenge test of G-CSF showed an increase in the platelet count and an augmentation of ADP- and collagen-induced platelet aggregation. In the use of G-CSF, patients who produce a rapid increase in platelet levels could be at greater risk for thrombotic events and need to be followed-up carefully.     

Acute arterial thrombosis due to platelet aggregation in a patient receiving granulocyte colony-stimulating factor

We describe a 44-year-old man with non-Hodgkin's lymphoma receiving granulocyte colony-stimulating factor (G-CSF) who developed an acute arterial thrombosis. The removed thrombus contained large amounts of platelet aggregation. A rapid increase of platelets and increased adenosine diphosphate (ADP)- and collagen-induced platelet aggregation were observed at the time of the thrombotic event. A challenge test of G-CSF showed an increase in the platelet count and an augmentation of ADP- and collagen-induced platelet aggregation. In the use of G-CSF, patients who produce a rapid increase in platelet levels could be at greater risk for thrombotic events and need to be followed-up carefully.     

Recombinant human granulocyte-macrophage colony-stimulating factor promotes megakaryocyte maturation in nonhuman primates

Megakaryocytes are responsive to several nonlineage-specific cytokines in vitro. In this study, we examined the in vivo effects of recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) on late stages of megakaryocytopoiesis in the rhesus monkey. Four rhesus monkeys were given 10 micrograms/kg body weight/day of rh GM-CSF s.c. in two divided doses daily for 8 days. Megakaryocyte maturation was evaluated serially by measuring nuclear ploidy and cytoplasmic size. GM-CSF-treated monkeys developed significant shifts in ploidy distribution from days 3 through 15 (p less than or equal to 0.001), with increased frequencies of 64N and 128N megakaryocytes. Mean megakaryocyte size increased 92.5% on day 9, paralleling the increase in DNA content, although megakaryocyte size within ploidy groups did not increase. Megakaryocyte number remained unchanged following rh GM-CSF treatment. The platelet count responses were variable, and mean platelet volume did not change. The present study demonstrates that therapeutic doses of rh GM-CSF stimulate megakaryocyte endomitosis and increase mean size. The data indicate that GM-CSF is effective in promoting the maturation stage of megakaryocyte development but does not result in a consistent thrombopoietic response.     

Long-term recombinant human granulocyte colony-stimulating factor (rhG-CSF) treatment severely depresses murine marrow erythropoiesis without causing an anemia.

We hereby report profound effects of long-term granulocyte colony-stimulating factor (G-CSF) administration on murine erythropoiesis. Recombinant human (rh)G-CSF (150 micrograms/kg body weight/day) was administered over 24 days to female C57Bl mice. Marrow erythroid colony-forming units (CFU-E) and erythroblast numbers declined to less than 5% of normal, whereas splenic erythropoiesis simultaneously increased. Splenic erythropoiesis effectively compensated for the loss of marrow erythropoiesis as indicated by the maintenance of a normal hematocrit. In the marrow the numbers of spleen colony-forming units (CFU-S) and erythroid burst-forming units (BFU-E) declined as well. Simultaneously, however, these numbers increased both in the spleen and in the peripheral blood by a factor of 20 to 30. These findings suggest a continuous migration of stem cells and progenitor cells out of the marrow and an efficient seeding in the spleen, directly or indirectly induced by G-CSF. In addition the differentiation and/or amplification of BFU-E to CFU-E was impaired in the marrow but not in the spleen. The marrow and splenic microenvironment also behaved differently with respect to granulopoiesis. G-CSF did not lead to an enhanced granuloid amplification in the spleen but exerted its proliferation activity mainly in the marrow. These findings imply that prolonged G-CSF treatment might cause erythroid depression in animals and humans when spleen erythropoiesis is less efficient than in mice.     

Mobilization of peripheral blood stem cells with chemotherapy and recombinant human granulocyte colony-stimulating factor (rhG-CSF): a randomized evaluation of different doses of rhG-CSF

To date, no randomized study has compared different doses of recombinant human granulocyte colony-stimulating factor (rhG-CSF) following submyeloablative mobilization chemotherapy. Therefore, we evaluated the effect of different doses of rhG-CSF following mobilization chemotherapy on yields of CD34+ peripheral blood stem cells (PBSC). Fifty patients were randomized to receive 8 (n = 25) versus 16 microg/kg/d (n = 25) of rhG-CSF following mobilization chemotherapy. The median number of CD34+ cells collected after 8 microg/kg/d of rhG-CSF was 2.36 x 10(6)/kg (range, 0.21-7.80), compared with 7.99 (2.76-14.89) after 16 microg/kg/d (P < 0.001). Twenty out of 25 (80%) patients in the low-dose and 23 out of 25 (92%) in the high-dose rhG-CSF arm underwent high-dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT). Median days to white blood cell engraftment in patients mobilized with 8 microg/kg and 16 microg/kg of rhG-CSF were 12 (10-20) and 9 (8-11) respectively (P < 0.001). There was no difference between the two groups regarding the other parameters of peritransplant morbidity: days to platelet engraftment (P = 0.10), number of red blood cell (P = 0.56) and platelet transfusions (P = 0.22), days of total parenteral nutrition requirement (P = 0.84), fever (P = 0.93) and antibiotics (P = 0.77), and number of different antibiotics used (P = 0.58). These data showed that higher doses of rhG-CSF following submyeloablative mobilization chemotherapy were associated with a clear dose-response effect based on the collected cell yields. Based on the parameters of peritransplant morbidity, 8 microg/kg/d was as effective as 16 microg/kg/d except for a rapid neutrophil engraftment in the high-dose arm. Therefore, in routine clinical practice, despite some advantage in the use of higher doses of rhG-CSF, lower doses may be used for PBSC collections following chemotherapy-based mobilization regimens in this cost-conscious era.     

Clinical application of recombinant human colony-stimulating factor (rhG-CSF) in bone marrow transplantation]

Human granulocyte colony-stimulating factor (G-CSF) specifically stimulates granulocyte production and enhances functions of mature granulocytes. It also proliferates myeloid leukemic cells. The molecule was purified and molecularly cloned in 1986. In this study, 51 patients received single daily injections with recombinant human (rh) G-CSF (2-20 micrograms/kg) after bone marrow transplantation were compared with control 36 patients. Blood neurophilic recovery was accelerated remarkably by rhG-CSF administration without any adverse effects including delay in reticulocyte and platelet recoveries. Furthermore, the duration for the management in laminar airflow room and febrile days greater than or equal to 38 degrees C were shortened in rhGS-CSF treated group. In 6 patients with myeloid leukemia in relapse, we also tested the effects of the rhG-CSF-combined conditioning regimen. In all the patients tested, the leukemic cells responded to rhG-CSF. Clinically no relapse was observed and 3 patients are well on day 224-357 for the time being. These findings indicate that rhG-CSF is very useful in bone marrow transplantation.     

Recombinant human granulocyte colony-stimulating factor (G-CSF) combined conditioning regimen for allogeneic bone marrow transplantation (BMT) in standard-risk myeloid leukemia

We previously suggested that using a combined conditioning regimen including rhG-CSF with allogeneic BMT in refractory AML and CML in blast crisis might reduce the rate of relapse and improve disease-free survival, without any major side effects. In this study, we used the same protocol for 10 AML patients in complete remission (CR) and 6 CML patients in the chronic phase (CP). We compared disease-free survival as well as toxic side effects of the regimen with 6 AML patients in CR and 6 CML patients in CP treated with chemoradiotherapy without G-CSF. The conditioning regimen consisted of TBI and high-dose AraC. RhG-CSF was infused continuously at a dose of 5 μg/kg/day, starting 24 hr before the initial dose of total body irradiation (TBI) until the end of AraC therapy. In all 28 cases, there were no early stage deaths due to regimen-related toxicity (RRT). None of the 10 AML cases treated with the G-CSF combined regime relapsed. In 6 AML cases treated conventionally without G-CSF, one patient died of infection and another relapsed. There were no relapses in either CML group. In the combined G-CSF group, one patient died of interstitial pneumonitis 48 days after BMT, while the rest of the CML cases are still alive. There were no relapses with rhG-CSF and no serious adverse effects in terms of RRT, acute graft vs. host disease (GVHD), or leukocyte recovery. Am. J. Hematol. 57:303–308, 1998. © 1998 Wiley-Liss, Inc.     

Recombinant human granulocyte colony-stimulating factor increases circulating CD34-positive cells in patients with AIDS

 In a gene therapy-based treatment of AIDS, it would be desirable to have as many transduced target cells as possible. A limiting factor is the number of target cells. In this study, we investigated whether it was possible to increase the absolute number of one possible target cell, i.e., the circulating hematopoietic progenitor cells (CD34 cells) in patients with AIDS, using the recombinant human granulocyte colony-stimulating factor (G-CSF). Eight patients with AIDS were treated with G-CSF for neutropenia (<1.0×10⁹/l). Treatment consisted of daily subcutaneous injections with 300 μg G-CSF for 3–5 consecutive days. Within 5 days of initiation of G-CSF therapy, an increase in the absolute neutrophil count (ANC) was seen in all patients. There was a median increase in ANC from 0.4 to 3.4×10⁹/l. In addition, G-CSF treatment significantly increased the absolute number of CD34 cells. The median increase in CD34 cells was from 0.8 to 2.2×10⁶/l. Finally, using a highly sensitive HIV-1 RNA PCR, we found that treatment of AIDS patients with G-CSF did not lead to enhanced HIV replication. These observations indicate that G-CSF may be used to mobilize CD34 cells in patients with AIDS, e.g., for a gene therapy protocol. Received: 8 August 1996 / Accepted: 27 January 1997     

Endotoxin down-modulates granulocyte colony-stimulating factor receptor (CD114) on human neutrophils

During infection, the development of nonresponsiveness to granulocyte colony-stimulating factor (G-CSF) may be influenced by the down-modulation of G-CSF receptor (G-CSFR) by cytokines. This down-modulation was studied during experimental human endotoxemia. Healthy volunteers received either 2 ng/kg endotoxin (lipopolysaccharide [LPS], n=20) or placebo (n=10) in a randomized, controlled trial. Endotoxin infusion increased the mean fluorescence intensity of the neutrophil activation marker CD11b >300% after 1 h (P<.001 vs. placebo). LPS infusion down-modulated G-CSFR expression in as early as 60 min (-17%; P=.001 vs. placebo). Down-modulation was almost maximal at 90 min and persisted for 6 h (-50% from baseline; P<.0001 vs. placebo). Plasma levels of G-CSF started to increase only after G-CSFR down-modulation had occurred and peaked 37-fold above baseline at 4 h (P<.0001 vs. placebo). In conclusion, LPS down-modulates G-CSFR expression in humans, which may render neutrophils less responsive to the effects of G-CSF and, thereby, compromise host defense mechanisms.     

Mechanism of synergy between granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor in colony formation from human marrow cells in vitro

The synergy of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and human granulocyte colony-stimulating factor (G-CSF) in the colony formation derived from human marrow cells was studied. The colony formation stimulated by GM-CSF plus G-CSF was dependent on the dose of each CSF, with the plateau for the number of GM colonies being higher than the sum of the individual plateaus by GM-CSF or G-CSF. Analysis of the colonies formed by GM-CSF plus G-CSF revealed efficient formation of neutrophil and monocyte colonies. To study the effect of GM-CSF and G-CSF on the maintenance of the progenitors that respond to the synergy of the CSFs, addition of each CSF to the medium of clonal cell culture was delayed. The progenitors that formed colonies on day 7 due to synergy of the CSFs were perfectly maintained by GM-CSF for at least 72 h and the progenitors that formed colonies on day 14 due to synergy of the CSFs were partly maintained by G-CSF or GM-CSF. The DNA synthetic rate of the progenitor cells that respond to GM-CSF plus G-CSF was significantly lower than those that respond to GM-CSF or G-CSF. According to light scatter analysis of phagocyte-depleted marrow mononuclear cells (PD-MMCs) using a flow cytometer, the peak population of progenitors that respond to GM-CSF plus G-CSF was in the smaller part of the PD-MMCs than those to GM-CSF or G-CSF. These results indicated that the progenitors to the synergy of GM-CSF and G-CSF are in a different proliferative state than those to each CSF. The synergy of GM-CSF and G-CSF depends on each CSF maintaining the viability of a different population of GM progenitors that can form GM colonies by both CSFs together.     

Recombinant human granulocyte colony-stimulating factor accelerates hematopoietic recovery after DLA-identical littermate marrow transplants in dogs.

We studied whether treatment of dogs with recombinant human granulocyte colony-stimulating factor (rhG-CSF), after 920 cGy total body irradiation (TBI) and transplantation of 3.3 +/- 1.0 x 10(8) bone marrow cells per kilogram from a DLA-identical littermate, accelerated hematopoietic recovery and influenced the incidence of subsequent marrow graft failure or graft-versus-host disease (GVHD). Ten animals were treated with 100 micrograms rhG-CSF/kg/d from days 1 through 10 after TBI. Results were compared with those of historical control of 14 dogs not administered rhG-CSF. Neither group of dogs received GVHD prophylaxis. The median time to recovery of 1,000 neutrophils/mm3 was 8 days for dogs administered rhG-CSF compared with 14 days in controls (logrank test: P less than .03). The median time to reach 100 monocytes/mm3 was 17 days in G-CSF-treated dogs compared with 49 days in controls (P less than .002). The median time to attain 500 lymphocytes/mm3 was 15 days versus 31 days, respectively (P less than .01). The median time to reach 20,000 platelets/mm3 was 26 versus 20 days (P = .68). Graft failure occurred in 1 of 10 G-CSF-treated dogs versus 2 of 14 controls (two-tailed Fisher's exact test: P = 1.00). GVHD was seen in 4 of 9 rhG-CSF-treated dogs compared with 1 of 12 controls (P = .12). Two G-CSF-treated dogs died of GVHD versus none of the controls (P = .17). No unusual toxicities were seen in dogs receiving rhG-CSF. In summary, rhG-CSF significantly accelerated recovery of neutrophils, monocytes, and lymphocytes after DLA-identical littermate marrow transplantation without altering platelet recovery. Graft failure was not seen more often than in controls, but there was a trend toward an increased incidence of GVHD.     

Recombinant human granulocyte colony-stimulating factor (rh-G-CSF) may accelerate hematopoietic recovery after HLA-identical sibling allogeneic peripheral blood stem cell transplantation

We studied the effects of recombinant human granulocyte colony-stimulating factor (G-CSF) on hematopoietic recovery and clinical outcome in patients undergoing allogeneic peripheral blood stem cell (PBSC) transplantation. Fifty-six patients with hematological malignancies who underwent allogeneic PBSC transplantation between 1995 and 1998 were entered into this study. Twenty-eight patients who received daily G-CSF from day +1 after allogeneic PBSC transplantation until the absolute neutrophil count (ANC) reached >0.5 x 10(9)/l for 3 consecutive days were compared with 28 patients (control group) who did not receive G-CSF in a non-randomized manner. The study group and the control group were comparable with respect to baseline patient and transplantation characteristics. Median times to ANC of >0.5 x 10(9)/l and 1 x 10(9)/l with or without G-CSF were 12 days (range 8-21), 13 days (10-32) (P = 0.04) and 13 days (9-21), 15 days (11-44) (P = 0.02), respectively. Median times to reach a platelet count of >20 x 10(9)/l with and without G-CSF were 11 days (0-20) and 13 days (9-26), respectively (P = 0.03). The incidence of febrile episodes was significantly lower with G-CSF, 75% vs 100% (P = 0.008). Patients receiving G-CSF had less grade III-IV mucositis than those who did not receive G-CSF (P = 0.01). There was also no increase in the incidence and severity of acute GVHD in patients using G-CSF (P = 0.22). Although the number of relapsing patients was greater in the G-CSF group (seven vs three patients), this was not statistically significant (P = 0.24). Disease-free and overall survival rates did not differ between the two groups (P = 0.58 and 0.53, respectively). The administration of G-CSF after allogeneic PBSC transplantation provided faster neutrophil and platelet engraftment associated with less severe mucositis and less febrile episodes.