Pre-B-Cell Colony Enhancing Factor (PBEF) Is a New Cytokine Regulating Myeloid Differentiation in Healthy Individuals and Patients with Severe Congenital Neutropenia (CN).
Authors: Skokowa Julia , Dan Lan , Cario Gunnar , Schambach Axel , Zeidler Cornelia , Baum Christopher , Stanulla Martin , Welte Karl 
Published: October 2019

Pre-B-cell colony-enhancing factor (PBEF) was first isolated from blood lymphocytes and was found to be involved in the maturation of B-cell precursors. Recently, PBEF was described as cytosolic enzyme nicotinamide phosphoribosyltranserase (NAmPRTase), promoted NAD+ biosynthesis. In this study we tested the ability of PBEF to induce myelopoiesis. Primary CD34+ cells treated with 50 ng/ml of PBEF in the serum-free RPMI medium without any additional cytokines, led to differentiation into mature granulocytes and macrophages, as assessed by FACS analysis (expression of CD11b, CD14, CD16), and by morphology. The same results were observed after lentiviral transduction of CD34+ progenitors with PBEF-GFP reporter. PBEF-dependent maturation was accompanied by increased mRNA expression of hematopoietic transcription factors such as C/EBPα , C/EBPε , PU.1, and ELA2. Moreover, PBEF had synergistic effect on G-CSF-triggered myeloid differentiation of CD34+ cells. Interestingly, G-CSF-treated differentiated CD34+ cells produced increased amounts of PBEF mRNA, as well as of intracellular and secreted PBEF protein in a time-dependent manner, as measured by real-time RT-PCR, ELISA, FACS analysis, and confocal microscopy. Increased synthesis of PBEF was in line with high NAD+ levels in G-CSF-treated cells. Expression of PBEF mRNA during myeloid differentiation was measured by laser-assisted single-cell picking and real-time quantitative RT-PCR analysis of different myeloid precursor cells (myeloblasts, promyelocytes, myelocytes, metamyelocytes, mature granulocytes and monocytes) from bone marrow smears of healthy individuals. PBEF mRNA expression was continually increased during myeloid differentiation with highest levels in mature granulocytes and monocytes.

In addition, we compared expression patterns of PBEF mRNA/protein in CD33+ progenitors and PMNs of healthy individuals after in vivo and in vitro treatment with G-CSF. We found PBEF mRNA/protein expression to be markedly increased in both cell types after G-CSF simulation (12 times for granulocytes, and 10 times for monocytes). Intriguingly, PBEF mRNA/protein expression in CD33+ cells and PMNs from G-CSF-treated patients with severe congenital neutropenia (CN) was also up-regulated and was significantly higher, as in G-CSF-treated healthy donors, suggesting that myeloid cells from CN patients try to overcome the differentiation arrest by upregulating PBEF. This was also supported by the fact that CN patients had at least 20 times higher PBEF plasma levels, in comparison to control group. G-CSF-dependent increase of PBEF expression was in line with increased NAD+ levels in G-CSF-treated cells. Indeed, in vitro PBEF treatment as well as lentiviral transduction of CD34+ cells from one CN patient with PBEF-GFP reporter gene resulted in partial restoration of myelopoiesis in vitro. Taken together, PBEF promotes maturation of myeloid cells by NAD+ dependent pathway. Moreover, increased expression of PBEF with subsequent up-regulation of NAD+ synthesis in CD33+ cells and neutrophils from CN patients could reveal mechanisms of G-CSF-dependent restoration of defective myelopoiesis in these patients.

rPBEF protein and anti-PBEF antibody were provided by Amgen, USA