Granulocyte colony-stimulating factor down-regulates the surface expression of the human leucocyte adhesion molecule-1 on human neutrophils in vitro and in vivo

Summary. The leucocyte adhesion molecule-1 (LAM-1) is the human homologue of the murine peripheral lymph node homing receptor, MEL-14, and might play a crucial role in neutrophil localization at inflammatory sites. We have reported previously that recombinant human granulocyte colony-stimulating factor (rhG-CSF) stimulates or enhances several neutrophil functions in vivo , as well as in vitro. To further explore the possible role of G-CSF in inflammation we studied the effect of rhG-CSF on the surface expression of LAM-1 on human neutrophils, both in vitro and in vivo. The expression of LAM-1 by human neutrophils was investigated by indirect immunofluorescence using flow cytometry and monoclonal antibodies anti-Leu-8 and TQ1. A whole blood analysis was performed to minimize in vitro manipulation. Most circulating human neutrophils expressed LAM-1 on the cell surface. Brief exposure of neutrophils to rhG-CSF in vitro decreased the surface expression of LAM-1. rhG-CSF down-regulated neutrophil LAM-1 expression in a time- and dose-dependent manner. Neutrophils from healthy volunteers and from patients who were receiving rhG-CSF exhibited a decreased expression of LAM-1 after rhG-CSF administration, and the expression thereafter returned or overshot the pretreatment level after stopping rhG-CSF administration. These findings indicate that rhG-CSF down-regulates the surface expression of LAM-1 on human neutrophils in vivo , as well as in vitro , and G-CSF might participate in neutrophil-endothelial cell interaction in inflamed tissue.     

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.     

Expression of adhesion molecules during apoptosis of circulating neutrophils in COPD

STUDY OBJECTIVES: Neutrophil accumulation occurs in the lungs of patients with COPD. This can be due to increased recruitment and/or delayed tissue clearance. Previous studies have described alterations in circulating neutrophils in these patients that can facilitate the former. Dysregulation of neutrophil apoptosis may contribute to the latter. This study investigated the potential abnormalities of the apoptotic process in COPD patients. DESIGN: Prospective study. SETTINGS: Outpatient clinic in a urban, tertiary hospital. PATIENTS: Fourteen stable patients with COPD, 8 smokers with normal lung function, and 8 healthy nonsmoking subjects. MEASUREMENTS AND RESULTS: We cultured circulating neutrophils that had been harvested from the study subjects at 2, 6, and 24 h. Apoptosis was assessed using flow cytometry by annexin binding and CD16 expression. The surface expression of the adhesion molecules Mac-1 (CD11b) and L-selectin (CD62L) also was determined by flow cytometry. The percentage of apoptotic neutrophils increased with time similarly in all groups. However, the surface expression of Mac-1 (CD11b) was higher, and that of L-selectin (CD62L) was lower, during apoptosis in the neutrophils of patients with COPD. CONCLUSIONS: These results show that, quantitatively, in vitro neutrophil apoptosis in COPD patients occurred at a rate similar to that found in healthy individuals and smokers with normal lung function. Qualitatively, however, the increased surface expression of Mac-1 (CD11b) and the decreased surface expression of L-selectin (CD62L) observed in the apoptotic neutrophils of COPD patients indicate increased activation during the apoptotic process. This may be relevant for the pathogenesis of COPD.     

Altered function and surface marker expression of neutrophils induced by rhG-CSF treatment in severe congenital neutropenia.

Neutrophils from patients suffering from severe congenital neutropenia (SCN), who were receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF), were investigated in order to analyze the previously described decrease in chemotaxis. This study demonstrated the decreased chemotaxis to five well-known chemoattractants, FMLP, C5a, IL-8, LTB4 and PAF. To further investigate this impairment of patients' neutrophils, receptors and receptor turnover for chemoattractants were examined using flow cytometry. We found 1) increased FMLP receptor and decreased C5a receptor expression, 2) a normal expression of intracellular FMLP receptors after incubation with PMA, 3) increased loss and decreased re-expression of FMLP receptors after incubation with this peptide, 4) normal expression of adhesion glycoproteins CR3 (CD11b/CD18) and LFA1 (CD11a/CD18), 5) further signs of in vivo preactivation: high expression of Fc gamma-RI (CD64) and Fc gamma-RII (CD32), decreased expression of Fc gamma-RIII (CD16), increased expression of CD14, and low expression of HLA-DR. These data demonstrate that the decrease of chemotaxis of neutrophils from SCN patients is not due: a) to a decrease in the number of intra- or extracellular FMLP receptors; b) to a decrease of adhesion molecules. However, the decreased chemotaxis could result from an altered FMLP receptor turnover. The relevance of the altered Fc gamma-receptor pattern for the in vivo occurrence of side-effects, e.g. the necrotic vasculitis, of G-CSF treatment is discussed.     

Autologous transplantation with peripheral blood mononuclear cells collected after administration of recombinant granulocyte stimulating factor

Peripheral blood mononuclear cells (PBMC) were collected after the administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and used as the sole source of hematopoietic stem cells after myeloablative therapy with busulfan (Bu) and cyclophosphamide (Cy). These studies were performed in 12 patients with malignancies (4 non-Hodgkin's lymphoma, 5 breast cancer, 1 testicular carcinoma, 1 Wilm's tumor, and 1 undifferentiated carcinoma) who had bone or bone marrow disease or had low marrow cellularity. rhG-CSF (16 micrograms/kg/d) was administered for 5 to 7 days by subcutaneous injection and PBMC were collected for 2 to 5 days beginning on day 4 after initiation of rhG-CSF, using continuous-flow blood-cell separators that processed 10 to 12 L of whole blood. From a median of three collections, a mean of 24.0 x 10(8) (+/- 10.5 SD) total nucleated cells/kg containing 12.6 x 10(8) (+/- 4.5 SD) mononuclear cells/kg, 7.3 x 10(6) (+/- 4.3 SD) CD34+ cells/kg and 20.5 x 10(4) (+/- 28.1 SD) granulocyte-macrophage colony-forming units (CFU-GM)/kg were harvested and cryopreserved. After the administration of Bu 14 to 17 mg/kg and Cy 120 to 150 mg/kg, PBMC were thawed and infused. One patient received rhG-CSF after the infusion of PBMC and the remaining 11 patients did not receive postinfusion growth factors. Mean days to recovery of neutrophil levels of 0.1, 0.5, and 1.0 x 10(9)/L were 11.4 (range, 9 to 13), 12.7 (range, 10 to 15), and 13.6 (range, 11 to 16) and the mean day to platelet transfusion independence was 13.3 (range, 7 to 49). Time to recovery of neutrophils to 0.5 and 1.0 x 10(9)/L and platelets to 20 x 10(9)/L was more rapid than in historical patients treated with Bu and Cy who received marrow alone or marrow followed by the posttransplant administration of rh-G or GM-CSF. No graft failures have been observed with a follow-up of 4 to 12 months. These results indicate that PBMC collected after rhG-CSF lead to rapid hematopoietic recovery after myeloablative chemotherapy.     

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.     

Ex vivo expanded peripheral blood progenitor cells provide rapid neutrophil recovery after high-dose chemotherapy in patients with breast cancer

Ex vivo expanded peripheral blood progenitor cells (PBPCs) have been proposed as a source of hematopoietic support to decrease or eliminate the period of neutropenia after high-dose chemotherapy. CD34 cells were selected from rhG-CSF mobilized PBPCs from patients with breast cancer and were cultured for 10 days in defined media containing 100 ng/mL each of rhSCF, rhG-CSF, and PEG-rhMGDF in 1 L Teflon bags at 20 000 cells/mL. After culture the cells were washed and reinfused on day 0 of transplantation. On day +1, cohort 1 patients (n = 10) also received an unexpanded CD34-selected PBPC product. These patients engrafted neutrophils (absolute neutrophil count, >500/microL) in a median of 6 (range, 5-14) days. Cohort 2 patients (n = 11), who received expanded PBPCs only, engrafted neutrophils in a median of 8 (range, 4-16) days. In comparison, the median time to neutrophil engraftment in a historical control group of patients (n = 100) was 9 days (range, 7-30 days). All surviving patients are now past the 15-month posttransplantation stage with no evidence of late graft failure. The total number of nucleated cells harvested after expansion culture was shown to be the best predictor of time to neutrophil engraftment, with all patients receiving more than 4 x 10(7) cells/kg, engrafting neutrophils by day 8. No significant effect on platelet recovery was observed in any patient. These data demonstrate that PBPCs expanded under the conditions defined can shorten the time to engraftment of neutrophils compared with historical controls and that the rate of engraftment is related to the dose of expanded cells transplanted.     

In vivo activation of human neutrophil functions by administration of recombinant human granulocyte colony-stimulating factor in patients with malignant lymphoma

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) was administered (50 to 800 micrograms/m2) once daily as a half-hour intravenous (IV) infusion for 14 days to seven patients with malignant lymphoma. In all patients, administration of rhG-CSF not only ameliorated the decrease in absolute neutrophil count after the cytotoxic chemotherapy but also enhanced superoxide (O2-) release in neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP). The priming effect of rhG-CSF on neutrophil O2- release was rapid (evident within 6.5 hours) and sustained at least for 24 hours after a single IV administration of rhG-CSF. The responsiveness to further in vitro challenge of rhG-CSF was lost or reduced in neutrophils isolated after rhG-CSF treatment, indicating that neutrophils already primed in vivo by rhG-CSF are desensitized to this factor. In contrast to the results obtained with FMLP, when phorbol myristate acetate (PMA) was used as stimulus, no consistent enhancement of O2- release was observed, suggesting that rhG-CSF modulates the signal transduction pathways linked to FMLP receptors rather than increases the components of the O2- producing enzyme complexes. Administration of rhG-CSF also rapidly (evident within 15 minutes) caused an increase in expression of neutrophil C3bi-receptors that was sustained for at least 24 hours after a single IV administration of rhG- CSF. Pharmacokinetic study of rhG-CSF showed a half-life (t1/2) of 114 min. These findings show that rhG-CSF is a potent activator for neutrophil functions both in vivo and in vitro.     

A successful case of congenital agranulocytosis treated with recombinant human granulocyte colony-stimulating factor]

A 1-month-old girl was admitted because of staphylococcal cellulitis of the buttock and the shoulder, and peripheral agranulocytosis. CBC on admission showed 10,100/microliters of WBC with 1% mature neutrophils, 5% monocytes, 6% eosinophils, 2% basophils and 86% lymphocytes. Bone marrow aspiration revealed maturation arrest of neutrophil precursors at the level of myelocyte. We treated the patient with subcutaneous rhG-CSF (Kirin-Amgen, Tokyo) for sequential 7-day course at the starting dose of 3 micrograms/kg, and increased weekly. The dose was escalated at the level of 18 micrograms/kg for 2 weeks subcutaneously, 8 days after effective dose of 18 micrograms/kg, the absolute neutrophil counts more than 1,000/microliters was attained, and bone marrow aspiration showed an increase of neutrophil precursors beyond the myelocyte level with maturation. Our case proved that both WBC and absolute neutrophil counts were increased parallel with the dose escalation of rhG-CSF. Shortly after the cessation of rhG-CSF, WBC and absolute neutrophil counts were decreased. No side effect was detected except for mild splenomegaly which was resolved after cessation of rhG-CSF. In methylcellulose culture with PHA-LCM, marrow cell of patient produced normal number of CFU-GM, and myeloid precursors could proliferate and differentiate to normal polymorphonuclear neutrophils, but rhG-CSF produced only small number of CFU-GM. Our case confirms that rhG-CSF is a new approach to control the life-threatening infection of congenital agranulocytosis.     

Clinical study of rhG-CSF (KRN8601) in patients with myelodysplastic syndrome]

A clinical study of rhG-CSF (KRN8601) in patients with myelodysplastic syndrome (MDS) was performed to investigate the hematopoietic effects and the increase of neutrophils. The rhG-CSF was administered daily by intravenous infusion over 30 min. to 21 patients with MDS (PARA = 11, RAEB = 4, RAEB in T = 6). The dose was escalated stepwise from 50 to 400 microgram/m2 every week. Within one week to 26 days after commencement of rhG-CSF administration, the increases of absolute neutrophil counts in peripheral blood were observed in all patients. Treatment with rhG-CSF enhanced normal marrow myeloid cell differentiation and maturation in 3 of 9 PARA patients and in 3 of 4 RAEB patients. None of patients changed to acute leukemia attributable to rhG-CSF, but one of RAEB patient and two of RAEB in T patients progressed to leukemic phase in 21 days or two months after treatment. Minor side effects or abnormal laboratory findings were observed in 3 patients (14.3%). These results suggested that treatment with rhG-CSF was well tolerated and effective for improving the neutropenia between 50 to 400 micrograms/m2 in patients with MDS.     

Effect of recombinant human granulocyte colony-stimulating factor on T-lymphocyte function and the mechanism of this effect

Whether recombinant human granulocyte colony-stimulating factor (rhG-CSF) affects lymphocyte function directly or indirectly is controversial. In this study, we found that T-cell proliferation was decreased considerably in response to phytohemagglutinin in donors who received rhG-CSF but was partly restored after monocytes were removed. Intracellular cytokine staining revealed that the interferon gamma-interleukin 4 ratio decreased by 5.97-fold in donor CD4+ cells after rhG-CSF treatment. No effect of rhG-CSF on ex vivo T-cell function was observed. rhG-CSF indirectly induced significant quantitative and qualitative changes on lymphocytes, including a decrease in T-cell proliferation and type 2 helper T-cell polarization of the cytokine profile. Although monocytes suppressed T-cell proliferation, the suppressive activity induced by the quantitative change in monocyte numbers cannot completely account for the hyporesponsiveness of T-lymphocytes. We believe that there must be another mediating factor. In addition, the numbers and mean fluorescence intensities of CD14CD86+ cells and CD19+CD80+ cells declined significantly in the peripheral blood after rhG-CSF treatment. Suboptimal amounts of stimulatory signals provided by low expression levels of B7 molecules on antigen-presenting cells (monocytes, B-lymphocytes) may help explain the alteration in T-cell proliferation. In addition, the absolute counts of CD3+CD4-CD8 cells in the peripheral blood were markedly increased and enriched in leukapheresis products following G-CSF treatment. These suppressor cells may contribute to T-cell hyporesponsiveness.     

Efficacy of recombinant human granulocyte colony-stimulating factor in a murine model of pneumococcal pneumonia: effects of lung inflammation and timing of treatment

The effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in a murine model of pneumococcal pneumonia was examined. Intranasal inoculations were 10(7) cfu/mouse (high inoculum) and 5 x 10(4) cfu/mouse (low inoculum) of Streptococcus pneumoniae, which induced severe or mild lung inflammation, respectively. With the low inoculum, rhG-CSF significantly improved survival when initiated 24 h or 10 min before, but not when initiated 24 h after, infection. Pretreatment with rhG-CSF significantly increased myeloperoxidase (MPO) activity in lungs 8 h after the infection and increased circulating neutrophil count 24, 48, and 72 h after infection. In contrast, rhG-CSF did not improve survival of animals infected with the high inoculum and did not increase MPO activity or neutrophil count in blood over those of sham-treated controls. These data strongly suggest that the severe inflammatory response typically observed in pneumococcal pneumonia recruits a maximum number of neutrophils in the lungs and thus masks the beneficial effect of rhG-CSF.     

Activation of peripheral and in vivo transmigrated neutrophils in patients with stable coronary artery disease

Accumulating evidence support a role of neutrophils in coronary artery disease (CAD). However little is known about the action of neutrophils at a local inflammatory site represented by an atherosclerotic plaque. To gain insight into these issues, we applied a skin blister model that permits analyses of in vivo transmigrated neutrophils. We hypothesised that the chronic inflammation in stable CAD mediates priming of neutrophils that impacts the out-come of neutrophil action at an inflammatory site. Thirteen patients with angiographically verified CAD were eligible for study entry together with 13 age and sex matched controls. Markers of inflammation (IL-6 and CRP), neutrophil activation (IL-8 and MMP-9/NGAL), and functional aspects (CD11b up-regulation and intracellular H(2)O(2) production) of peripheral and in vivo transmigrated neutrophils were studied. Systemic IL-8 and MMP-9/NGAL concentrations were significantly increased in patients indicating a primed state in circulating neutrophils. In vivo transmigrated neutrophils in stable CAD patients had an increased propensity to release MMP-9/NGAL and a reduced capacity to up-regulate CD11b and to produce hydrogen peroxide. These aberrations at the inflammatory site may be a consequence of a primed state of circulating neutrophils and point towards potential mechanisms whereby neutrophils at a local inflammatory site may contribute to the pathogenesis of CAD.     

In vivo neutrophil and lymphocyte function studies in a patient with leukocyte adhesion deficiency type II

We investigated in vivo neutrophil and lymphocyte function in a patient who lacks Sialyl-Lewis-X, a ligand for the selectin family of leukocyte adhesion molecules (leukocyte adhesion deficiency II, LAD II). As assessed by skin chamber and skin window techniques, in vivo chemotaxis of neutrophils was markedly impaired (less than 6% of normal values). A marginal pool was present as determined by an increase in circulating neutrophils after epinephrine injection and calculated recovery of infused radiolabeled autologous neutrophils. Kinetic studies showed a reduced half-life of 3.2 hours (normal 7 hours) and markedly increased turnover rate (cells/kg/d) of approximately eight times the normal value. A normal antibody response to the T-cell-dependent antigen bacteriophage phi X174 showed that T/B-cell interaction is not affected in LAD II. These findings provide direct evidence that the selectin family and its ligands play an important role in neutrophil function.     

Mobilization and collection of PBSC in healthy donors: comparison between two schemes of rhG-CSF administration

Abstract: Procurement of a high number of progenitor cells is of primary interest in allogeneic PBSC transplantation. We have retrospectively compared toxicity, mobilization effect and progenitor cell yields of two different rhG-CSF schedules in 11 consecutive healthy individuals donating their PBSC. Five of them received rhG-CSF 16 μg/kg/d for 4 subsequent d in 2 divided subcutaneous injections (group A); similarly, 6 donors received rhG-CSF 10 μg/kg/d for 5 d (group B). The aphereses were started the last day of rhG-CSF treatment; 9 donors underwent 2 aphereses, one underwent 1 and another 3 procedures, always on subsequent days. Toxicity was mild, but moderate thrombocytopenia developed following apheretic collections, irrespective of rhG-CSF schedule. In all the donors WBC, as well as circulating CD34⁺ cells, CFU-GM, CFU-GEMM and BFU-E dramatically increased over the baseline values, peaking on d 5 or 6, with no statistical difference between the 2 groups for the height of the cell peaks. Also the peripheral lymphoid cell populations (CD3⁺, CD19⁺ and CD56⁺/CD3^-) increased following the rhG-CSF administration. The number of MNC, CFU-GM, BFU-E, CFU-GEMM, as well as CD34⁺, CD3⁺, CD19⁺ and CD56⁺/CD3 cells collected by apheresis showed no statistical difference in the 2 groups. Overall, 8 of the 11 donors collected the target number of CD34⁺ cells > 4times10⁶/kg ideal recipient body weight with the first apheresis, with no difference between the 2 groups. Mobilization with rhG-CSF in healthy donors enables the collection of large number of progenitor cells with modest side effects. A schedule of 10 μg/kg for 5 d is as effective as 16 μg/kg for 4 d. A single apheresis would be enough in 80% of cases.     

Paradoxical drop in circulating neutrophil count following granulocyte-colony stimulating factor and stem cell factor administration in rhesus macaques

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) is frequently used therapeutically to treat chronic or transient neutropenia and to mobilize hematopoietic stem cells. Shortly following G-CSF administration, we observed a dramatic transient drop in circulating neutrophil number. This article characterizes this effect in a rhesus macaque animal model. METHODS: Hematologic changes were monitored following subcutaneous (SQ) administration of G-CSF. G-CSF was administered as a single SQ dose at 10 microg/kg or 50 microg/kg. It was also administered (10 microg/kg) in combination with stem cell factor (SCF; 200 microg/kg) over 5 days. Flow cytometry was performed on serial blood samples to detect changes in cell surface adhesion protein expression. RESULTS: Neutrophil count dramatically declined 30 minutes after G-CSF administration. This decline was observed whether 10 microg/kg G-CSF was administered in combination with SCF over 5 days, or given as a single 10 microg/kg dose. At a single 50 microg/kg dose, the decline accelerated to 15 minutes. Neutrophil count returned to baseline after 120 minutes and rapidly increased thereafter. An increase in CD11a and CD49d expression coincided with the drop in neutrophil count. CONCLUSION: A transient paradoxical decline in neutrophil count was observed following administration of G-CSF either alone or in combination with SCF. This decline accelerated with the administration of a higher dose of G-CSF and was associated with an increase in CD11a and CD49d expression. It remains to be determined whether this decline in circulating neutrophils is associated with an increase in endothelial margination and/or entrance into extravascular compartments.     

In vivo synergy between recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in baboons enhanced circulation of progenitor cells

Recombinant human stem cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) are synergistic in vitro in stimulating the proliferation of hematopoietic progenitor cells and their precursors. We examined the in vivo synergy of rhSCF with rhG-CSF for stimulating hematopoiesis in vivo in baboons. Administration of low-dose (LD) rhSCF (25 micrograms/kg) alone did not stimulate changes in circulating WBCs. In comparison, administration of LD rhSCF in combination with rhG-CSF at 10 micrograms/kg or 100 micrograms/kg stimulated increases in circulating WBCs of multiple types up to twofold higher than was stimulated by administration of the same dose of rhG-CSF alone. When the dose of rhG-CSF is increased to 250 micrograms/kg, the administration of LD rhSCF does not further increase the circulating WBC counts. Administration of LD rhSCF in combination with rhG-CSF also stimulated increased circulation of hematopoietic progenitors. LD rhSCF alone stimulated less of an increase in circulating progenitors, per milliliter of blood, than did administration of rhG-CSF alone at 100 micrograms/kg. Baboons administered LD rhSCF together with rhG-CSF at 10, 100, or 250 micrograms/kg had 3.5- to 16-fold higher numbers per milliliter of blood of progenitors cells of multiple types, including colony-forming units granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming and burst-forming units-megakaryocyte (CFU- MK and BFU-MK) compared with animals given the same dose of rhG-CSF without rhSCF, regardless of the rhG-CSF dose. The increased circulation of progenitor cells stimulated by the combination of rhSCF plus rhG-CSF was not necessarily directly related to the increase in WBCs, as this effect on peripheral blood progenitors was observed even at an rhG-CSF dose of 250 micrograms/kg, where coadministration of LD rhSCF did not further increase WBC counts. Administration of very-low- dose rhSCF (2.5 micrograms/kg) with rhG-CSF, 10 micrograms/kg, did not stimulate increases in circulating WBCs, but did increase the number of megakaryocyte progenitor cells in blood compared with rhG-CSF alone. LD rhSCF administered alone for 7 days before rhG-CSF did not result in increased levels of circulating WBCs or progenitors compared with rhG- CSF alone. Thus, the synergistic effects of rhSCF with rhG-CSF were both dose- and time-dependent. The doses of rhSCF used in these studies have been tolerated in vivo in humans.(ABSTRACT TRUNCATED AT 400 WORDS)     

The use of rh-G-CSF in chronic autoimmune neutropenia: reversal of autoimmune phenomena, a case history

An 8-year-old boy had been suffering from chronic autoimmune neutropenia for more than 5 years. The neutropenia proved to be resistant to high-dose steroids and intravenous (either low- or high-dose) immunoglobulin (Ig) therapy. The chronic autoimmune thrombocytopenia and recurrent phases of autoimmune haemolytic anaemia did, however, respond to high-dose prednisone. Other signs of immune dysregulation in this patient consisted of insulin-dependent diabetes mellitus type I (IDDM) and an acquired hypogammaglobulinaemia, most compatible with common variable immunodeficiency (CVI). Prior to rhG-CSF therapy the child had suffered for more than 2 years from recurrent life-threatening bacterial infections.
Anti-neutrophil autoantibodies had pan-FcγRIII (CD16, NA1/NA2) specificity. The neutropenia as well as the anti-neutrophil autoantibodies disappeared when subcutaneous rhG-CSF therapy was started. Upon tapering rhG-CSF, anti-FcγRIII antibodies reappeared together with an absolute neutropenia. Renewed administration resulted again in the normalization of symptoms.
Soluble FcγRIII (sFcγRIII) antigen levels in plasma increased dramatically during rhG-CSF treatment. These high levels of sFcγRIII together with increased numbers as well as decreased apoptotic reactions of neutrophils apparently result in adsorption of the autoantibodies in vivo , contributing to the normalization of autoimmune-mediated neutropenia upon rhG-CSF treatment. Long-term administration of rhG-CSF represents an alternative in the treatment of autoimmune neutropenia.     

Pharmacokinetics and adverse events following 5-day repeated administration of lenograstim, a recombinant human granulocyte colony-stimulating factor, in healthy subjects

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) (lenograstim) was administered to healthy subjects at doses of 2, 5 and 10 micrograms/kg/day for 5 days (twice a day subcutaneously) to examine the optimal dose and schedule of lenograstim in mobilizing peripheral blood progenitor cells (PBSC) for allogeneic transplantation. Lenograstim administration significantly increased CD34+ cells in a dose-related manner. A significant correlation was observed between the maximal post-dosing counts and the pre-dosing baseline counts of CD34+ cells. Peripheral neutrophils increased markedly by seven to 13 times from the baseline to a peak of approximately 40,000/microliter on day 5 for the 5 and 10 micrograms/kg/day doses. After peak serum concentration (Cmax) was attained 4 h following administration, serum G-CSF declined with time in a log-linear fashion. The Cmax and 12 h area-under-the-curve increased dose dependently, but minimum drug level increased up to day 2 and then decreased until day 5. Clearance decreased with increasing dosage at the first dose, and increased significantly at the last dose. We found a highly significant correlation between absolute neutrophil counts and clearance for each dose. Adverse events most frequently occurred on day 6, with increases of alkaline phosphatase and lactate dehydrogenase and onset of bone pain. Increases of aspartate aminotransferase and alanine aminotransferase occurred as delayed events. Platelet count gradually decreased after the end of drug administration to 57% of the pre-dosing count on day 10, but was still within the normal range. These preliminary results suggest that repeated doses of lenograstim induce mobilization of PBSC in a dose-dependent manner and the pre-dosing baseline count of PBSC may predict the post-dosing maximal mobilization. The drug treatment may cause delayed-onset moderate thrombocytopenia and increased transaminase, and the drug clearance changes in a complex manner during repeated dosing.     

In vitro and in vivo hematopoietic effect of mutant human granulocyte colony-stimulating factor.

About 100 derivatives of human recombinant granulocyte colony-stimulating factor (rhG-CSF) were created by various gene-mutagenic techniques, and KW-2228, in which amino acids were replaced at five positions of N-terminal region of intact rhG-CSF, was picked up and evaluated for its biologic and physicochemical properties in comparison with intact rhG-CSF. KW-2228 showed two to four times higher specific activity than that of intact rhG-CSF in mouse and/or human bone marrow progenitor cells by colony-forming unit assay in soft agar, and by cell-proliferation assay in liquid culture. KW-2228 showed a potency to increase peripheral neutrophil counts when it was administered to normal C3H/He mice by single intravenous injection. Increase of total leukocyte count and neutrophils was observed, with peak level at 8 to 12 hours at low doses (0.5 to 1.0 micrograms/mouse), and the highest level was maintained for 24 to 30 hours at high doses (5 to 10 micrograms/mouse). The granulopoietic effect of KW-2228 was examined by several doses of single course (once daily for 10 days) or multiple courses (twice daily injection for 5 days followed by cessation for 9 days on one cycle, 3 cycles in total) of treatment. KW-2228 showed higher activity than that of rhG-CSF, especially at sub-optimal doses of multiple courses of treatment. Furthermore, KW-2228 was found to be more stable physicochemically and biologically than intact rhG-CSF, especially under thermal conditions at 56 degrees C and in the human plasma at 37 degrees C, suggesting a protease resistancy. Pharmacokinetic study showed that plasma concentration of KW-2228 assayed for its bioactivity maintained a higher level than that of intact rhG-CSF for 60 minutes after intravenous injection of this protein to normal mice. Those results suggest that KW-2228 might show a superior in vivo hematopoietic effect to intact rhG-CSF due to its high specific activity to progenitor cells, and also due to its improved physicochemical, biologic, and pharmacokinetic stability in host animals.