Administration of Granulocyte Colony-Stimulating Factor Does Not Restore Defective Expression of bcl-2 and bcl-2-xL in Myeloid Progenitor Cells of Patients with Severe Congenital Neutropenia (Kostmann Syndrome).
Authors: Cario Gunnar , Skokowa Julia , Wang Zheng , Bucan Vesna , Zeidler Cornelia , Stanulla Martin , Schrappe Martin , Welte Karl 
Published: October 2019

Abstract
Severe congenital neutropenia (SCN; Kostmann syndrome) is characterized by a maturation block in the myelopoiesis at the promyelocytic/myelocytic stage leading to decreased amounts of neutrophils in bone marrow and peripheral blood. This maturation arrest was previously associated with an accelerated apoptosis. Administration of recombinant human granulocyte colony-stimulating factor (G-CSF) sufficiently increases neutrophil numbers in most patients. Recently, it was demonstrated that the elevated degree of apoptosis was accompanied by a selective decreased expression of the anti-apoptotic Bcl-2 protein in myeloid progenitor cells in 4 surviving members of the original “Kostmann family” and 1 patient with SCN of unknown inheritance (Carlsson et al., Blood2004; 103: 3355–3361). In these patients, it was shown that the administration of G-CSF restored Bcl-2 expression and corrected the abnormal acceleration of apoptosis. To test whether this observation is a common feature of SCN patients myeloid progenitor cells, we analyzed the mRNA and protein expression of bcl-2 in CD33+ bone marrow myeloid progenitor cells from 6 G-CSF treated SCN patients not related to the original “Kostmann family” in comparison to that of 2 long-term G-CSF treated patients with neutropenia other than SCN and healthy controls without and after administration of G-CSF (4 and 3, respectively). Additionally, the mRNA expression of the Bcl-2-related genes, bcl-xL (anti-apoptotic) and bax (pro-apoptotic), and the expression of caspase 9, an important intracellular amplifier of apoptotic signaling was investigated. Gene expression was measured by quantitative real-time PCR, and protein expression by immunofluorescence and confocal microscopy. We observed a significant increase of bcl-2 expression after administration of G-CSF in CD33+ cells of healthy controls (fold change (FC) = 1.7, p = 0.0011, t-test). In contrast, bcl-2 was significantly lower expressed in CD33+ cells from long-term G-CSF treated SCN patients as compared to that of long-term G-CSF treated patients with neutropenia other than SCN (FC = 20, p = 0.015, t-test), and healthy controls without (FC = 7, p < 0.0001) and after administration of G-CSF (FC = 13, p < 0.0001). The expression of bcl-xL did show a similar pattern with a significant difference comparing SCN patients and healthy controls without G-CSF (FC = 4.4, p = 0.018). In contrast, the expression of caspase 9 was significantly upregulated in CD33+ cells of SCN patients as well as G-CSF treated controls compared to healthy controls without G-CSF administration (FC = 8.9, p = 0.002). Bax was similarly expressed in all groups. The expression pattern of bcl-2 was confirmed on the protein level. In summary, the expression of bcl-2 was defective in SCN patients as it was described by Carlsson et al., and a similar pattern was observed for bcl-xL. In contrast to published data, long-time administration of G-CSF did not normalize the expression of both anti-apoptotic Bcl-2 family members in our patients. We conclude, that a mechanism other than the restoration of Bcl-2 and Bcl-xL expression is responsible for the increase of neutrophils after G-CSF treatment in our patient sample.