Gene expression analysis illuminates the transcriptional programs underlying the functional activity of ex vivo-expanded granulocytes

Abstract

Global gene expression analysis established the temporal expression patterns and programs underlying the development of functional activity of ex vivo-expanded (EXE) human granulocytes, as well as differences compared with peripheral blood (PB) granulocytes. CD34+progenitor cells were cultured for 3 wk to induce rapid expansion and granulocytic differentiation, with 40% CD15+cells by day 3 and 90% by day 12. Phagocytic and respiratory burst activity increased with the fraction of CD15++CD11b+cells (myelocytes to segmented) and peaked by day 17. However, only 25% of CD15++CD11b+cells were phagocytic, and respiratory burst activity was one-third that of PB granulocytes. EXE granulocytes from later days and PB granulocytes showed similar expression of Fcγ receptors (-1A, -2A, -2C, -3A) and complement receptors (-1, -3, -4). Later downregulation of CD36 (expressed by macrophages) suggests lineage plasticity early in granulocytic differentiation. Expression in mature EXE and PB granulocytes was similar for most Fcγ receptor-mediated phagocytosis signaling proteins, including high-level expression of Hck, Fgr, and the actin-related protein 2/3 complex. Lower expression of Lyn, Cdc42, pleckstrin, and PKCβIby EXE granulocytes may explain decreased phagocytosis. PB and mature EXE granulocytes expressed similar levels of NADPH oxidase complex genes and receptors for fMLP-mediated respiratory burst. Lower burst activity by EXE granulocytes may result from lower expression of Raf1 and PKCζ. Elevated expression of toll-like receptor (TLR)2, TLR1, and CD14 in mature EXE and PB granulocytes supports a role for the TLR2 and CD14 pathway in zymosan-mediated respiratory burst activity. Lower activity in EXE granulocytes may be due to greater expression of IRAK3, which inhibits TLR-mediated signaling.