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Annals of Clinical & Laboratory Science, vol. 38, no. 4, 2008
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 Abstract. 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) Introduction Granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage-CSF (GM-CSF) are 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
[email protected].
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 CD34positive immature bone marrow cells to mature peripheral granulocytes [3], but are not expressed in lymphocytes, normoblasts, or basophils [4].
0091-7370/08/0400-0331. $2.45. © 2008 by the Association of Clinical Scientists, Inc.
332 Annals of Clinical & Laboratory Science, vol. 38, no. 4, 2008 Chronic myeloid leukemia (CML) is a myeloproliferative 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 factorindependent 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. Materials and Methods 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.
Results 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 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). Group CML (n = 47) Control (n = 25)
G-CSF (pg/ml)
GM-CSF (pg/ml)
21.14 ± 2.99 19.38 ± 2.93
2.50 ± 0.51 2.43 ± 0.42
G-CSF/GM-CSF and receptors in CML 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). Receptor
Group
Neutrophils
Monocytes
G-CSFr
CML (n = 47) Control (n = 25)
1224 ± 94* 1820 ± 129
1098 ± 109 1172 ± 241
GM-CSFr
CML (n = 47) Control (n = 25)
862 ± 57 832 ± 101
2805 ± 343 3343 ± 330
*p
