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

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 PKCβ_I by 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.