To characterize the transcriptional program that governs terminal granulocytic differentiation in vivo, we performed comprehensive microarray analyses of human bone marrow populations highly enriched in promyelocytes (PMs), myelocytes/ metamyelocytes (MYs), and neutrophils (bm-PMNs). These analyses identified 11 310 genes involved in differentiation, of which 6700 were differentially regulated, including previously unidentified effector proteins and surface receptors of neutrophils. Differentiation of PMs toward MYs was accompanied by a marked decline of proliferative and general cellular activity as defined by down-regulation of E2 promoter binding factor (E2F) target genes; cyclin dependent kinases 2, 4, and 6; and various metabolic, proteasomal, and mitochondrial genes. Expression patterns of apoptosis genes indicated death control by the p53 pathway in PMs and by death receptor pathways in bm-PMNs. Effector proteins critical for host defense were expressed successively throughout granulocytic differentiation, whereas receptors and receptor ligands essential for the activation of the host defense program were terminally up-regulated in bm-PMNs. The up-regulation of ligandreceptor pairs, which are defined inducers as well as target genes of nuclear factor-B (NF-B), suggests a constitutive activation of NF-B in bm-PMNs by autocrine loops. Overall IntroductionPolymorphonuclear neutrophilic granulocytes (neutrophils/PMNs) constitute the most abundant population of white blood cells and are essential players in innate immune defense of mammalian hosts against microorganisms. Once neutrophils have migrated to sites of infection, they recognize microorganisms and their products to initiate a first line of defense using a number of distinct mechanisms. These defense mechanisms include phagocytosis, generation of reactive oxygen intermediates, and the release of antimicrobial granule proteins for killing and degradation of microorganisms. 1,2 Neutrophils are short-lived cells, which are continuously generated from hematopoietic stem cells (HSCs) in the bone marrow (BM) by a process called granulopoiesis. The hallmark of early granulopoiesis is the successive commitment of pluripotent HSCs via multipotent common myeloid progenitors (CMPs) and bipotent granulocyte-macrophage progenitors (GMPs) toward unipotent progenitors restricted to the granulocytic lineage. 3,4 Once the progenitors are committed to the granulocytic lineage, they initiate terminal granulopoiesis and differentiate into mature neutrophils. Terminal granulopoiesis gives rise to a series of morphologically distinct stages, which are readily identified by their characteristic nuclear shape and their content of granules. At the myeloblast/ promyelocyte (MB/PM) stages the cells still proliferate and generate primary granules with their constituting proteins. At the myelocyte/metamyelocyte (MC/MM) stages, cell proliferation and expression of primary granule proteins stop concomitantly with the successive generation of secondary and tertiary granules and the...

OLFM4 was identified initially as a gene highly induced in myeloid stem cells by G-CSF treatment. A bioinformatics method using a global meta-analysis of microarray data predicted that OLFM4 would be associated with specific granules in human neutrophils. Subcellular fractionation of peripheral blood neutrophils demonstrated complete colocalization of OLFM4 with the specific granule protein NGAL, and stimulation of neutrophils with PMA resulted in corelease of NGAL and OLFM4, proving that OLFM4 is a genuine constituent of neutrophil-specific granules. In accordance with this, OLFM4 mRNA peaked at the MY/MM stage of maturation. OLFM4 was, however, present in only 20-25% of peripheral blood neutrophils, as determined by immunocytochemistry and flow cytometry, whereas mRNA for OLFM4 was present in all MY/MM, indicating post-transcriptional regulation as a basis for the heterogeneous expression of OLFM4 protein.

Arginase 1 (ARG1) metabolizes arginine, thus reducing the availability of arginine as a substrate for nitric oxide synthase (NOS). The decreased production of nitric oxide (NO) by NOS and the production of ornithine by ARG1 affect immune responses and tissue regeneration at sites of infection, respectively. We here demonstrate that ARG1 is synthesized in myelocytes/metamyelocytes and is stored in gelatinase granules. In accordance with this, activated neutrophils coreleased ARG1 and gelatinase to the extracellular environment on stimulation with phorbol-12-myristate 13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), or tumor necrosis factor ␣ (TNF-␣). Overall, these findings define ARG1 as a genuine gelatinase granule protein and support a model in which activated neutrophils release ARG1 at sites of infection to modulate immune responses and promote tissue regeneration. (Blood.

Ficolins are soluble molecules that bind carbohydrate present on the surface of microorganisms and function as recognition molecules in the lectin complement pathway. Three ficolins have been identified in humans: ficolin-1, ficolin-2, and ficolin-3. Ficolin-1 is synthesized in monocytes and type II alveolar epithelial cells. Ficolin-1 has been shown to be present in secretory granules of human neutrophils, but it is not known which subset of the neutrophils' secretory granules harbors ficolin-1. To determine the exact subcellular localization of ficolin-1 in neutrophils, recombinant ficolin-1 was expressed in Chinese hamster ovary cells and used for generation of polyclonal antibodies. This allowed detection of ficolin-1 in subcellular fractions of human neutrophils by ELISA, by Western blotting, and by immunohistochemistry. Real-time PCR examination of normal human bone marrow showed FCN1 gene expression largely in myelocytes, metamyelocytes, and band cells with a profile quite similar to that of gelatinase. In accordance with this, biosynthesis studies of neutrophils precursor cells showed that ficolin-1 was primarily synthesized in myelocytes, metamyelocytes, and band cells. Immunohistochemistry and subcellular fractionation demonstrated that ficolin-1 is primarily localized in gelatinase granules but also in highly exocytosable gelatinase-poor granules, not described previously. Ficolin-1 is released from neutrophil granules by stimulation with fMLP or PMA, and the majority becomes associated with the surface membrane of the cells and can be detected by flow cytometry. Our studies show that neutrophils are a major source of ficolin-1, which can be readily exocytosed by stimulation.