Model analysis of the contrasting effects of GM-CSF and G-CSF treatment on peripheral blood neutrophils observed in three patients with childhood-onset cyclic neutropenia

Human cyclic neutropenia (CN) is a rare haematological disorder characterized by regular fluctuation in the serial count of blood neutrophils. The oscillations occur at subnormal levels with a stable 3-week period. To reduce the risk of serious infections during the severe neutropenic nadir phases (<0.25 ×10⁹ neutrophils/l) patients are usually treated with recombinant growth factors. Compared with G-CSF, which has been shown to enhance the amplitudes substantially, the response to GM-CSF is poor: neutrophil numbers are not amplified, the cycles remain unchanged or are dampened. However, two cases with a modest neutrophil increase have been reported in the literature. In a recently published clinical study the different effects of GM-CSF and G-CSF application have been investigated in the same patients. Based on a mathematical model of CN we previously proposed, the detailed neutrophil data measured in this study are analysed by simulation. The contrasting clinical results can be quantitatively explained by the model concept of regulatory control together with possible individual feedback defects, i.e. abnormally reduced mitotic responsiveness of granulopoietic progenitor cells to GM-CSF and G-CSF.     

Acquired cyclic neutropenia: Successful treatment with prednisone

A previously healthy woman developed severe, periodic neutropenia after ingestion of phenylbutazone. Oscillations in the monocyte count and hemoglobin concentrations also occurred. The neutropenic episodes were associated with severe bacterial infections requiring hospitalization. Lithium induced a transient interruption in the neutropenia, but continued use was ineffective. Prednisone in a dosage of 100 mg daily successfully interrupted the neutrophil cycling and prevented infection. The patient has remained in remission on 10 mg of prednisone on alternate days.     

Serum G-CSF levels are not increased in patients with antibody-induced neutropenia unless they are suffering from infectious diseases

By the use of a G-CSF-specific ELISA we determined the serum granulocyte-colony stimulating factor (G-CSF) levels in 63 patients with antibody-induced neutropenia including neonatal immune neutropenia, autoimmune neutropenia, and drug-induced immune neutropenia. In the sera of 20 patients, elevated G-CSF levels of 60-1006 pg/ml (normal <39 pg/ml) were observed. These patients suffered from infectious diseases at the time of blood collection. G-CSF levels normalized after successful antibiotic treatment, indicating that increased G-CSF production in patients with immune neutropenia may be primarily the result of infection and not of neutropenia.     

Response of T-beta CD8+ lymphocytosis-associated neutropenia to G-CSF

SUMMARY. Only limited data are reported on the response to granulocyte-colony stimulating factor (G-CSF) of large granular lymphocytosis-associated neutropenia. We report features of such a case who developed a suppurating leg ulcer following a dog bite. G-CSF was used to increase the neutrophil count, allowing the ulcer to be successfully skin grafted.     

Myelodysplasia and acute myeloid leukaemia in cases of aplastic anaemia and congenital neutropenia following G-CSF administration

Summary. Myelodysplasia and acute myeloid leukaemia (MDS/AML) developed in three cases of severe aplastic anaemia (SAA) and one case of congenital neutropenia (CN, Kostmann's disease) who received recombinant human granulocyte colony-stimulating factor (G-CSF) are reported. In these four MDS/AML cases, age at diagnosis of SAA/CN was 0–13 years, the cumulative dose of G-CSF was 98 μg/kg to 10 mg/kg over 1–57 months, and the interval from initiation of G-CSF to MDS/AML was 25, 23, 31 and 57 months, respectively. These results suggest a link between SAA/CN and MDS/AML in relation to G-CSF administration; however, large studies are necessary to determine if such a risk is significant in patients with SAA/CN who are treated with G-CSF.     

G-CSF receptor (CSF3R) mutations in X-linked neutropenia evolving to acute myeloid leukemia or myelodysplasia

X-linked neutropenia (XLN) is a rare form of Congenital Neutropenia (CN) caused by inherited gain-of-function mutations of WAS. Here we report 2 cases of the original L270P X-linked neutropenia kindred that evolved to MDS or AML, with acquisition of G-CSFR (CSF3R) mutations and monosomy 7. Thus, leukemic transformation with acquisition of CSF3R mutations and monosomy 7 is not restricted to classical congenital neutropenia with autosomal inheritance, but can also occur in other genotypes of inherited neutropenia.     

Hereditary cyclic neutropenia in the male members of a Chinese family with inverted Y chromosome

We describe four male members of a Chinese family, including the father and three sons, with hereditary cyclic neutropenia. These patients had all developed cyclic neutropenia in childhood with a cycle of around 21 d. Recurrent mucosa and skin infections with fever had occurred frequently, but gradually decreased in severity on reaching adulthood. Monocytosis was found during the neutrophil nadirs in all four patients. Mildly increased serum immunoglobulin (Ig)A and IgG levels, low levels of serum stem cell factor, as well as decreased sperm count and motility were demonstrated in the two elder sons. Chromosomal analysis showed a pericentric inversion of Y chromosome [46,X, inv(Y)(p11.2;q11.23)] in all of the men. These findings may suggest an association between cyclic neutropenia with oligospermia and inv(Y)(p11.2;q11.23) in this particular family.     

Long-term safety of treatment with recombinant human granulocyte colony-stimulating factor (r-metHuG-CSF) in patients with severe congenital neutropenias

Summary . Congenital neutropenias include a heterogenous group of diseases characterized by a decrease in circulating neutrophils. In phase I/II/III studies in patients with severe congenital and cyclic neutropenia, treatment with recombinant human granulocyte colony-stimulating factor (r-metHuG-CSF) resulted in a rise in the absolute neutrophil counts (ANC) and a reduction in infections. We report the effects of long-term safety of subcutaneous r-metHuG-CSF administration in 54 patients (congenital n = 44, cyclic n = 10) treated for 4–6 years. A sustained ANC response was seen in 40/44 severe congenital neutropenia patients and 10/10 cyclic neutropenia patients. Two patients required an increase of > 25% in dose to maintain a clinical response; one patient became refractory to therapy. A significant decrease in the incidence of severe infections and the need for intravenous antibiotics was noted. Significant adverse events noted which may or may not be related to therapy included: osteopenia ( n = 15), splenomegaly ( n = 12), hypersplenism ( n = 1), vasculitis ( n = 2), glomerulonephritis ( n = 1), BM fibrosis ( n = 2), MDS/leukaemia ( n = 3), and transient inverted chromosome 5q with excess blasts ( n = 1). R-metHuG-CSF has been well tolerated in the majority of patients and resulted in a longterm improvement in their clinical status.     

Alteration of colony-stimulating factor output, endotoxemia, and granulopoiesis in cyclic neutropenia.

Cellular and humoral factors involved in the regulation of granulopoiesis were evaluated in two patients with cyclic neutropenia by utilizing the agar-gel marrow culture technique to serially study marrow granulocytic colony-forming capacity (CFC) and the urinary output of colony-stimulating factor (CSF). CSF output varied inversely with peripheral neutrophil counts and directly with monocyte counts and evidence for infection (endotoxemia and/or staphylococcal abscesses). Following autologous infusion of one patient's plasma obtained during a period of neutropenia, increased urinary excretion of CSF occurred concomitant with increments in both marrow CFC and the proportion of granulocytic progenitor cells in DNA synthesis. Neutrophil periodicity was not altered by the administration of the neutropenic plasma. These findings are consistent with the hypothesis that cyclic neutropenia is caused by a quantitatively decreased entry of stem cells or granulocytic progenitor cells into granulopoiesis.     

Childhood onset cyclic neutropenia: G-CSF therapy restores neutrophil count but does not influence superoxide anion and cytokine release by neutrophils

Summary. In this paper we describe the case of a 16-year-old boy with childhood onset cyclic neutropenia (CN) with a 21d cycle who was successfully treated with recombinant granulocyte-colony stimulating factor (G-CSF). Cyclic therapy with G-CSF (5μg/kg/d s.c. for 1 week every 21d) maintained peripheral neutrophil count above the normal range, reduced the incidence of severe infections and significantly improved the patient's performance status throughout an 18-month follow-up. Phenotypic analysis of circulating lymphocytes demonstrated that G-CSF treatment does not modify the phenotypic profile of circulating B. T and NK cell populations. Circulating neutrophils released normal amounts of cytokines (including IL-1/3, IL-8, TNFα) and superoxide anion during G-CSF therapy.     

A Novel Mutation in the Juxtamembrane Intracellular Sequence of the Granulocyte Colony-Stimulating Factor (G-CSF) Receptor Gene in a Patient with Severe Congenital Neutropenia Augments G-CSF Proliferation Activity but Not through the MAP Kinase Cascade

We analyzed the structure of the granulocyte colony-stimulating factor (G-CSF) receptor gene in a 6-year-old female patient with severe congenital neutropenia (SCN) who experienced severe recurrent infections since 1 month of age. There is no family history of any similar disease. When the patient was 4 months old, she began receiving treatment with recombinant human G-CSF that resulted in a small increase in the neutrophil count sufficient for the prevention and treatment of bacterial infection. An analysis of complementary DNA for the patient's G-CSF receptor revealed a 3-base pair deletion in the juxtamembrane intracellular sequence. This deletion at the beginning of exon 16 was thought to be caused by alternative splicing; analysis of the DNA revealed a G-to-A point mutation of the final nucleotide of intron 15. To evaluate the functional activity of the G-CSF receptor with this 3-base pair deletion of the juxtamembrane region, we transfected this G-CSF receptor mutant into an interleukin 3-dependent cell line, BAF/3. BAF/3 cells expressing the mutant G-CSF receptor showed augmented proliferation activity in response to G-CSF compared with cells having the wild-type G-CSF receptor. Although the proliferation signal of G-CSF in normal hematopoiesis is transduced through the activation of MAP kinases, this G-CSF receptor mutant showed decreased activation of ERKI/2 in response to G-CSF compared with the wild type, but the transduced sig-nal for Stat3 activation by G-CSF was of the same magnitude as that of the wild-type G-CSF receptor. This result means that the augmented proliferation activity in response to G-CSF that we observed in cells having the G-CSF receptor gene with the 3-base pair deletion is transduced through an intracellular signaling pathway other than MAP kinase. Because SCN patients with a mutation in the G-CSF receptor frequently develop leukemia, this 3-base pair deletion in the juxtamembrane sequence of the G-CSF receptor gene in this patient may be one step in the course of leukemic transformation.     

Changes in light-scatter profile, membrane depolarization and calcium mobilization of neutrophils induced by G-CSF in vivo

Summary. To extend our studies about phenotypical and functional alterations of G-CSF-induced neutrophils we have evaluated their light-scatter profile, mobilization of intracellular calcium ([Ca²⁺]i) and membrane depolarization after stimulation. A significant increase in the forward scatter signals could be demonstrated in such neutrophils from patients with neutropenias of various origin and from healthy test subjects. This increase began 4 h and returned to normal 96 h after G-CSF injection in the latter group. We found an impairment of [Ca²⁺]i mobilization in neutrophils from patients with glycogen storage disease type IB after stimulation of these cells with fMLP, It was even more pronounced than in severe congenital neutropenia (SCN). However, [Ca²⁺]i fluxes were normal when ionomycin was used. Neutrophils from patients with cyclic neutropenia (cyNP) and chemotherapy-induced neutropenia (chNP) mobilized [Ca²⁺]i similar to those from healthy donors. Furthermore, we found a decreased percentage of neutrophils depolarizing after stimulation with fMLP and PMA in patients with SCN, whereas membrane depolarization was normal in patients with chNP and cyNP. All the alterations found here are suggested to be caused by a partial immaturity of the neutrophils, although in vivo activation and a direct effect of G-CSF on myeloid precursors might be involved.     

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.     

Acute myeloid/NK precursor cell leukemia with trisomy 4 and a novel point mutation in the extracellular domain of the G-CSF receptor in a patient with chronic idiopathic neutropenia

Chronic idiopathic neutropenia (CIN) has been well recognized as a granulocytic disorder not associated with increased risk to malignant transformation. Four cases, however, of acute myeloid leukemia have been recently reported in patients with CIN. In the current paper, we report on a CIN patient who developed acute myeloid/natural killer (NK) precursor cell leukemia 11 years after diagnosis and 4 months after initiation of treatment with recombinant human granulocyte colony-stimulating factor (rhG-CSF). Leukemic cells had trisomy 4 as the sole cytogenetic abnormality and, also, a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSFR) leading to truncated protein with a loss of 36 amino acids. There was no evidence that this receptor transmitted signals even in the presence of high doses of rhG-CSF in the cultures. We consider that CIN may be a preleukemic condition, at least in a subset of patients, and that rhG-CSF administration is unlikely to be involved in the leukemic transformation in this patient, although such a possibility could not be completely ruled out.     

G-CSF enhanced granulocyte production in a child with severe neutropenia in the setting of fatal virus-associated hemophagocytic syndrome.

A 14-year-old boy with fatal varicella zoster virus-associated hemophagocytic syndrome (VAHS) was treated with recombinant human granulocyte colony-stimulating factor (G-CSF) based on the finding that the patient had severe neutropenia and possible bacterial superinfection. Support for the G-CSF therapy in VAHS is provided by the recent reports that G-CSF is relatively specific for the granulocyte lineage; it would not activate mature monocyte/macrophage/histiocyte lineage in VAHS, where the most striking morphologic feature is histiocytic hyperplasia with hemophagocytosis. He responded well to G-CSF with an elevation of neutrophil counts. There were no effects on other blood cells. The result indicates that G-CSF is useful to increase granulocyte production in severe neutropenia, even in the setting of fatal VAHS.     

Adult-onset cyclic neutropenia responsive to cyclosporine therapy in a patient with ankylosing spondylitis

A 45-year-old female with a long history of HLA-B27-positive ankylosing spondylitis and ulcerative colitis developed cyclic neutropenia. She was hospitalized for high fever during each of three consecutive episodes of absolute neutropenia. On the third hospitalization, granulocyte-colony-stimulating factor (G-CSF), 5 μg/kg/day, was given by subcutaneous injection and resulted in an increase of absolute neutrophil count from 0 to 2.2 × 10⁹/liter and an associated decrease of platelet count and hemoglobin as well as severe bone and joint pain predominantly in the middle and lower back and purulent diarrhea. The back pain necessitated discontinuation of the drug. Oral cyclosporine therapy was begun, and although the neutrophil count continued to oscillate, both the peaks and the nadirs were higher than previously, and symptoms of neutropenia subsided. We conclude that cyclosporine can be an effective treatment for cyclic neutropenia associated with autoimmunity since G-CSF may cause exacerbations of autoimmune disorders.     

Bone mineralization and turnover in children with congenital neutropenia, and its relationship to treatment with recombinant human granulocyte-colony stimulating factor

Bone mineral content (BMC) of the radius was measured using single photon absorptiometry (SPA) in nine children with congenital neutropenia. Five had normal values. Two children with severe congenital neutropenia (SCN) had low BMC, and two boys with Schwachman syndrome had biochemistry suggestive of rickets.     

In vitro functions of neutrophils induced by treatment with rhG-CSF in severe congenital neutropenia.

Neutrophils (and monocytes) from 5 patients suffering from severe congenital neutropenia (SCN) were investigated in vitro after induction of this cell type by treatment with recombinant human granulocyte colony-stimulating factor (rh G-CSF) in vivo. Some abnormal morphological features were seen, such as anomalies of nuclei of phagocytes. No limitations were found 1) in the ability of neutrophils and monocytes to produce reactive oxygen intermediates (ROI) following stimulation with phorbol-myristate-acetate (PMA), 2) in the ability of neutrophils to phagocytose particles of zymosan A and to produce ROI simultaneously and 3) in the ability to kill bacteria of the species staphylococcus aureus. However the specific migration of neutrophils in a gradient of FMLP under soft agar was decreased to approximately 50% in all patients tested as compared to healthy controls. In addition, spontaneous motility was decreased in one patient. Nevertheless, the good clinical improvements of patients suffering from SCN after treatment with rh G-CSF appeared to be due to induction of neutrophils displaying overall good functional activities with respect to natural defense.