Annals of Clinical & Laboratory Science 38:331-337 (2008)
© 2008 Association of Clinical Scientists
G-CSF and GM-CSF Concentrations and Receptor Expression in Peripheral Blood Leukemic Cells from Patients with Chronic Myelogenous Leukemia
Jehoon Lee,
Yonggoo Kim,
Jihyang Lim,
Myungshin Kim and
Kyungja Han
Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea
Address correspondence to Kyungja Han, M.D., Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, 62 Yeouido-dong Yeongdeungpo-gu, Seoul, 150-713, Korea (South); tel 82 2 3779 1297; fax 82 2 3779 2285; e-mail hankja{at}catholic.ac.kr.
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Abstract
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Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-CSF (GM-CSF) are the principal cytokines in granulopoiesis and differentiation of granulocytic precursors. Their physiologic effects are mediated by binding to specific cell surface receptors (G-CSFr and GM-CSFr, respectively), which are widely expressed from immature bone marrow cells to mature peripheral granulocytes. The fact that concentrations of plasma G-CSF and GM-CSF and their receptors are changed in infectious diseases showing neutrophilia is known, but such changes in patients with chronic myelogenous leukemia (CML) have not been studied. Based on quantitative assays of plasma G-CSF and GM-CSF and their receptors on the peripheral granulocytes of CML patients and healthy controls, this study analyzes the differences between these groups in G-CSF and GM-CSF levels, as well as quantitative and qualitative changes in the receptors. Plasma levels of G-CSF and GM-CSF were measured in 47 patients in the chronic phase of CML and 25 healthy adults as normal controls. G-CSFr and GM-CSFr on peripheral granulocytes were analyzed by quantitative flow cytometry, and changes in G-CSF and GM-CSF receptor counts were also measured. Plasma concentrations of G-CSF and GM-CSF in CML patients were similar to normal controls (p >0.05). The quantity of G-CSFr on neutrophils was more highly expressed than on other cell types in both groups, and the amount of this receptor in patients with CML was less than in normal controls (p = 0.001). GM-CSFr was expressed in higher concentrations on monocytes than neutrophils, and there was no difference in the amount of GM-CSFr on neutrophils. After incubation with excess G-CSF, the expressed amounts of G-CSFr on neutrophils and monocytes were decreased in both groups. However, G-CSFr on the monocytes was decreased in healthy controls (p = 0.02) with no difference in patients with CML. The quantities of GM-CSFr expression on neutrophils and monocytes after incubation with excess GM-CSF were decreased in both groups. Granulocyte counts in peripheral blood of CML patients were not correlated with the plasma concentrations of G-CSF or GM-CSF, nor with the expression of G-CSFr or GM-CSFr on granulocytes. Granulopoiesis in patients with CML was not mediated by increased plasma CSF concentrations, and there was no difference in the amounts of G-CSFr or GM-CSFr expressed on the granulocytes. This suggests that a structural change may occur on monocytes of CML patients, since the binding capacity of G-CSFr to G-CSF on the monocytes is different from the normal controls.
Keywords: Granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), G-CSF receptor, GM-CSF receptor, chronic myelogenous leukemia (CML)
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Introduction
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Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-CSF (GM-CSF) are the principal cytokines in granulopoiesis and differentiation of normal bone marrow granulocytic precursors [1]. Their physiologic effects are mediated through binding to specific cell surface receptors [2]. These receptors (G-CSFr and GM-CSFr, respectively) are widely expressed from CD34-positive immature bone marrow cells to mature peripheral granulocytes [3], but are not expressed in lymphocytes, normoblasts, or basophils [4].
Chronic myeloid leukemia (CML) is a myelo-proliferative disease characterized by excessive clonal production of maturing myeloid cells. The molecular events that cause the disordered proliferation and differentiation, as well as clonal expansion of hematopoietic cells characteristic of CML, are poorly understood. However, evidence strongly suggests that the bcr/abl fusion gene product is involved in leukemogenesis [5]. The fusion of the first exon of abl with the first exon of bcr leads to relocation of the fusion protein to the cytoplasm with markedly increased tyrosine kinase activity [6]. Therefore, the fusion gene can induce cell proliferation and transformation of immature hematopoietic cells and prolong the growth factor-independent survival of CML progenitors by inhibition of apoptosis [7,8].
A defined region of the cytoplasmic domain of the G-CSFr transmits signals for maturation or differentiation of myeloid progenitor cells. Mutations in this region have been found in some patients with severe congenital neutropenia [9,10]. However, the possible alterations in the expressed levels of CSFr and in their binding affinities to plasma G-CSF or GM-CSF in patients with CML have not been studied.
In this study, the levels of plasma G-CSF and GM-CSF in patients in the chronic phase of CML were measured, and the expressed levels of G-CSFr and GM-CSFr were analyzed using a newly-devised quantitative flow cytometric assay. These levels were compared to those of normal granulocytes in peripheral blood. We also evaluated the binding capacities of CSFr to CSFs to demonstrate the roles of these cytokines in granulopoiesis.
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Materials and Methods
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Patients and healthy controls.
EDTA-anticoagulated peripheral blood samples were obtained from 47 patients (33 male, 14 female) with chronic phase CML who were cared for at the Catholic University St. Mary’s Hospital between May and December 2006, and from 25 healthy adults (14 male, 11 female) with no history of hematologic or inflammatory diseases. All samples were analyzed within 4 hr of collection and kept at room temperature (18–20°C) until analysis. The plasma specimens for quantitative determination of G-CSF and GM-CSF concentrations were separated immediately after collection of venous blood and stored in a –70°C freezer until analysis.
Quantitative determination of plasma G-CSF and GM-CSF concentrations.
For determination of G-CSF and GM-CSF concentrations in plasma, immunoassay kits (R & D Systems, Minneapolis, MN, USA) were used according to the manufacturer’s instructions. The assays employ the quantitative sandwich enzyme immunoassay technique. All samples and standards were assayed in duplicate and the average of each optical density was plotted on a standard curve to determine the concentrations.
Quantitative analysis of G-CSFr and GM-CSFr.
Each 50 µl sample of EDTA-treated whole blood was mixed with 5 µl of anti-G-CSFr antibody (phyco-erythrin[PE]-conjugated mouse anti-human CD114, PharMingen International, San Diego, CA, USA), or anti-GM-CSFr antibody (PE-conjugated mouse anti-human CD 116, Serotec, Oxford, UK). The samples were incubated in a dark room for 30 min. The erythrocytes were then lysed with 2 ml of lysing solution (Becton-Dickinson, Franklin Lakes, NJ, USA) and the sediments were washed in PBS. Fluorescence was analyzed in duplicate by flow cytometry (FACS Calibur, Becton-Dickinson) using CELLQuest software. Results were recorded as the geometric mean of gated cells. The mean number of bound PE molecules per cell was calculated using QuantiBRITE and QuantiQuest programs (Becton-Dickinson).
CSFr activity without binding to CSFs after incubation with excess amount of CSFs.
To estimate the functional binding activity of CSFr, leukocytes were incubated with excess amounts of CSFs for 1 hr at 37°C. After 3 washes with PBS, flow cytometry of CSFr using monoclonal antibodies was performed as described above. G-CSF (Neutrogen, Choong Wae Pharm, Korea) was added at a concentration of 0.5 µg/106 cells and GM-CSF (Leukogen, LG Pham, Korea) was added at a concentration of 2 µg/106 cells.
Statistics.
The data were analyzed using the Mann-Whitney U test. The significance was evaluated by the Wilcoxon signed ranks test using SPSS software. Using paired simple t-test, the amount of CSFr after binding to CSF was compared to the initial concentration. To test correlations among the amount of CSFr, plasma CSF concentration, and leukocyte count, the Pearson correlation coefficients and p values were calculated.
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Results
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Plasma concentrations of G-CSF were 19.38 ± 2.93 pg/ml in healthy controls and 21.14 ± 2.99 pg/ml in patients with CML (Table 1
), and there was no statistically significant difference (p = 0.70). The plasma concentrations of GM-CSF were also not statistically different in healthy controls (2.43 ± 0.42 pg/ml) and patients with CML (2.50 ± 0.51pg/ ml) (p = 0.74).
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Table 1. Plasma concentration of colony stimulating factors (G-CSF & GM-CSF) in healthy adults (control) and patients with chronic myelogenous leukemia (CML) (mean ± SD).
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G-CSFr was expressed in larger quantities on neutrophils than monocytes in both healthy controls and patients with CML (Table 2). In patients with CML, the quantity of G-CSFr on neutrophils was less than healthy controls (p = 0.001), but there was no difference on monocytes. GM-CSFr expression was stronger on monocytes than neutrophils in both groups, but the amounts of G-CSFr on neutrophils and monocytes were not significantly different between healthy controls and patients with CML.
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Table 2. Quantities of receptor molecules for colony stimulating factors (G-CSFr and GM-CSFr) on neutrophils and monocytes in patients with chronic myelogenous leukemia (CML) and healthy adults (control) (mean ± SD).
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After incubation with an excess amount of G-CSF, the amount of expression of G-CSFr on neutrophils was decreased in healthy adults and patients with CML (p = 0.001 in both groups) (Fig. 1, Table 3). The expressed quantity of G-CSFr on monocytes after binding with G-CSF was decreased in healthy adults (p = 0.02), but not in patients with CML (p = 0.54). After incubation with an excess amount of GM-CSF, the amount of expression of GM-CSFr on neutrophils and monocytes was decreased in healthy adults and patients with CML (p = 0.001 in both groups).
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Fig. 1. Dot scattergram of leukocytes in chronic myelogenous leukemia showing neutrophil gate (R1) and monocyte gate (R2) by flow cytometry. Histograms of neutrophils stained with anti-G-CSFr in excess amount of G-CSF showed less fluorescence intensity after incubation in G-CSF than before incubation. However, the histograms of monocytes in excess amount of G-CSF showed similar fluorescence intensity before (P1) and after (P2) incubation.
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Table 3. Quantities of receptor molecules for colony stimulating factors (CSFr) after CSF binding in healthy adults (control) and CML patients (number of bound PE molecules per cell ± SD).
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Analysis of correlations among leukocyte counts, plasma CSF concentrations, and expressed amounts of CSFr in patients with CML showed that G-CSFr on neutrophils was positively correlated with the expressed amounts of GM-CSFr on neutrophils, and G-CSFr or GM-CSFr on monocytes (Table 4). A positive correlation between plasma G-CSF and GM-CSF concentrations was evident, but there was no significant correlation between neutrophil counts and CSFs on neutrophils or monocytes.
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Table 4. Simple correlations among leukocyte counts, quantity of CSFr, and concentration of plasma CSF in CML patients. *p <0.05.G-CSFr and GM-CSFr are not expressed on lymphocytes, and neutrophils express more G-CSFr than GM-CSFr. However, the monocytes express more GM-CSFr than G-CSF [4]. These differences were found in patients with CML in this study. The smaller amount of G-CSFr expression on neutrophils, when compared to the healthy controls, may result from contamination of immature leukocytes in patients with CML.G-CSFr-deficient mice have chronic neutropenia as in G-CSF-deficient mice [17], but do not have the expected neutrophilia after administration of chemoattractant [18]. Point mutations in the G-CSFr gene were reported in two patients with severe congenital neutropenia, and these abnormal receptors interfered with the functions of normal receptors by means of a dominant negative mechanism [19]. These mutations were detected in patients who developed acute leukemia. This means that G-CSFr is important not only in the induction of neutrophilia, but also in the function of neutrophils. In this study, we evaluated the binding capacities of CSFr to CSF based on the characteristics of the receptors, which do not bind to antibodies after incubation with appropriate CSFs. The binding capacity of G-CSFr on monocytes to G-CSF in patients with CML was less than that of healthy controls, but the binding capacities of G-CSFr to G-CSF on neutrophils were not different in either group. These findings indicate the change in G-CSFr is specific for monocytes.Aberrant sialylation of CML granulocytes alters the binding of GM-CSF to GM-CSFr, and may also alter signal transduction, which in turn may be responsible for the immature phenotype of CML granulocytes [20]. In this study, the binding capacities of GM-CSFr to GM-CSF were decreased after binding with GM-CSF in healthy controls and patients with CML.Analysis of simple correlations among leukocyte counts, plasma CSF concentrations, and expression amounts of CSFr in patients with CML showed that G-CSFr on neutrophils was positively correlated with expressed amounts of GM-CSFr on neutrophils, and G-CSFr or GM-CSFr on monocytes. A positive correlation between plasma G-CSF and GM-CSF concentrations was also shown. However, there was no significant correlation between neutrophil counts and CSFs on neutrophils or monocytes.In conclusion, the expressed level of G-CSFr on neutrophils in patients with CML was lower than in healthy adult controls, but the plasma concentrations of CSFs related to neutrophilia were not increased. It is possible that patients with CML have less functional GM-CSFr than healthy adults. These findings suggest that lower levels of plasma CSFs concentration and dysfunction of CSFr, especially GM-CSF, in patients with CML are important points in discriminating the pathogenesis of neutrophilia from that of infection, and may play an important role in explaining leukemogenesis in patients with CML.
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Discussion
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The presence of G-CSFr on the gated leukemic blasts from newly diagnosed patients with acute leukemia or crisis of CML has been investigated using flow cytometric detection [3,11]. These studies suggest that the distribution of G-CSFr on leukemic cells possessing myeloid characteristics may be related to their maturation process [12]. However, quantitative assay of those receptors in patients with CML has not been reported.
In patients with CML, the quantity of G-CSFr in neutrophils was less than those of normal adults, and the G-CSFr was expressed in the greatest quantity in neutrophils, followed by the amount on monocytes. However, the amount of GM-CSFr on monocytes was greater than on neutrophils. These findings differ from the results from cases of infections [4], in which the amounts of CSFr revealed no significant differences between the infection groups.
The G-CSF treatment widely used in infectious disease has been reported to up-regulate G-CSFr in neutrophils [13], especially when associated with neutropenia [9,14]. The plasma level of G-CSF in CML patients after bone marrow transplantation positively correlates with engraftment [15]. It is important to realize that the growth factor has multiple effects on cells of the granulocytic lineage. This factor not only stimulates the proliferation and differentiation of myeloid precursors, but also enhances the functional activation of mature neutrophils [3,16]. In this study, the plasma concentrations of G-CSF and GM-CSF in patients with CML were similar to those of the healthy controls. This is a meaningful finding when contrasted to the results in cases of infection showing leukocytosis [4]. Our research indicates that leukocytosis in CML is not mediated by increasing G-CSF or GM-CSF.
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Acknowledgements
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We thank Professor Won-Il Kim for mentoring and interest in the study. This work was supported by the Clinical Research Foundation of Our Lady of Mercy Hospital in Incheon, Korea.
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References
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Abstract
Introduction
