Olfactomedin 4 defines a subset of human neutrophils.

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.

Alpha-1-antitrypsin is produced by human neutrophil granulocytes and their precursors and liberated during granule exocytosis.

Alpha-1-antitrypsin (A1AT) is an important inhibitor of neutrophil proteases including elastase, cathepsin G, and proteinase 3. Transcription profiling data suggest that A1AT is expressed by human neutrophil granulocytes during all developmental stages. A1AT has hitherto only been found associated with azurophile granules in neutrophils indicative of A1AT expression being restricted to the promyelocyte stage. We examined the localization and production of A1AT in healthy donor neutrophils and found A1AT to be a constituent of all granule subtypes and to be released from neutrophils following stimulation. A1AT is produced at all stages of myeloid maturation in the bone marrow. The production increases as neutrophils enter circulation and increases further upon migration to tissues as observed in skin windows and when blood neutrophils are incubated with granulocyte colony-stimulating factor. Neutrophils from patients with A1AT-deficiency carrying the (PI)ZZ mutation in the A1AT gene appeared structurally and functionally normal, but A1AT produced in leukocytes of these patients lacked the ability to bind proteases efficiently. We conclude that A1AT generation and release from neutrophils add significantly to the antiprotease levels in tissues during inflammation. Impaired binding of neutrophil A1AT to serine proteases in patients with (PI)ZZ mutations may enhance their susceptibility to the development of emphysema.

Ficolin-1 is present in a highly mobilizable subset of human neutrophil granules and associates with the cell surface after stimulation with fMLP.

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.

The secretory leukocyte protease inhibitor (SLPI) and the secondary granule protein lactoferrin are synthesized in myelocytes, colocalize in subcellular fractions of neutrophils, and are coreleased by activated neutrophils.

The secretory leukocyte protease inhibitor (SLPI) re-establishes homeostasis at sites of infection by virtue of its ability to exert antimicrobial activity, to suppress LPS-induced cellular immune responses, and to reduce tissue damage through inhibition of serine proteases released by polymorphonuclear neutrophil granulocytes (PMNs). Microarray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/metamyelocytes (MYs), and BM neutrophils demonstrates a transient, high mRNA expression of SLPI and genuine secondary granule proteins (GPs) in MYs. Consistent with this finding, immunostaining of BM cells showed SLPI and the secondary GP lactoferrin (LF) to be present in cells from the myelocyte stage and throughout neutrophil differentiation. Subcellular fractionation studies demonstrated the colocalization of SLPI and LF in subcellular fractions highly enriched in secondary granules. Finally, exocytosis studies demonstrated a corelease of SLPI and LF within minutes of activation. Collectively, these findings strongly indicate that SLPI is localized in secondary granules of PMNs. However, the amount of SLPI detected in PMNs is low compared with primary keratinocytes stimulated by growth factors involved in wound healing. This implicates that neutrophil-derived SLPI might not contribute essentially to re-establishment of homeostasis at sites of infection but rather, exert physiologically relevant intracellular activities. These might include the protection of secondary GPs against proteolytic activation and/or degradation by proteases, which might be dislocated to secondary granules at minute amounts as a consequence of spillover.

Arginase 1 is expressed in myelocytes/metamyelocytes and localized in gelatinase granules of human neutrophils.

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 alpha (TNF-alpha). 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.

Haptoglobin is synthesized during granulocyte differentiation, stored in specific granules, and released by neutrophils in response to activation.

Haptoglobin (Hp) is a plasma protein synthesized primarily by hepatocytes. It exerts a broad range of anti-inflammatory activities and acts indirectly as a bacteriostatic agent and an antioxidant by virtue of its ability to bind free hemoglobin (Hb) and to facilitate its immediate clearance by macrophages. We identified Hp as a novel specific granule protein of neutrophils by means of immunoelectron microscopy, subcellular fractionation, and exocytosis studies. Consistent with these findings, blood cells from a patient with specific granule deficiency (SGD) lacked neutrophil-derived Hp. Neutrophils contained a large amount of highly glycosylated Hp (beta-chain 45-65 kDa) synthesized in neutrophil precursors and stored in specific granules and a small amount of Hp (beta-chain 39 kDa) endocytosed from plasma and stored in secretory vesicles. Subsequent binding studies revealed that Hp from specific granules binds to Hb. Finally, the CCAAT enhancer binding protein-epsilon (C/EBPepsilon) induced Hp transcription in a myeloid cell line, suggesting that Hp expression in myeloid cells, as in hepatocytes, is at least partially regulated by members of the C/EBP transcription factor family. Collectively, these findings demonstrate that Hp is stored in specific granules and is released by neutrophils in response to activation. Hence, neutrophil-derived Hp might reduce tissue damage and bacterial growth at sites of infection or injury by propagating anti-inflammatory activities and Hb clearance.

Highly glycosylated alpha1-acid glycoprotein is synthesized in myelocytes, stored in secondary granules, and released by activated neutrophils.

Alpha-1-acid glycoprotein (AGP) is an acute-phase protein produced by hepatocytes and secreted into plasma in response to infection/injury. We recently assessed the transcriptional program of terminal granulocytic differentiation by microarray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/metamyelocytes (MYs), and BM neutrophils. These analyses demonstrated a transient, high mRNA expression of genuine secondary/tertiary granule proteins and AGP in MYs. In agreement with this, immunocytochemistry revealed the presence of AGP protein and the secondary granule protein lactoferrin in cells from the MY stage and throughout granulocytic differentiation. Immunoelectron microscopy demonstrated the colocalization of AGP and lactoferrin in secondary granules of neutrophils. This finding was substantiated by the failure to detect AGP and lactoferrin in blood cells from a patient with secondary/tertiary (specific) granule deficiency. In addition, Western blot analysis of subcellular fractions isolated from neutrophils revealed that neutrophil-derived AGP, localized in secondary granules, was abundant and highly glycosylated compared with endocytosed, plasma-derived AGP localized in secretory vesicles. Exocytosis studies further demonstrated a marked release of AGP and lactoferrin by activated neutrophils. Finally, induction of CCAAT/enhancer-binding protein (C/EBP)-epsilon in a myeloid cell line was shown to increase AGP transcript levels, indicating that AGP expression in myeloid cells, like in hepatocytes, is partially regulated by members of the C/EBP family. Overall, these findings define AGP as a genuine secondary granule protein of neutrophils. Hence, neutrophils, which constitute the first line of defense, are likely to serve as the primary local source of AGP at sites of infection or injury.