ALBERT

Description: IL-17 is a proinflammatory cytokine synthesized by a newly discovered subset of helper T-cells called TH17. In our search for targets that are regulated by IL-17, we have found that the gene for the bacteriostatic protein neutrophil gelatinase associated lipocalin (NGAL) is strongly induced in epithelial cells by this cytokine. NGAL is a siderophore-binding protein that binds bacterial catecholate-type ferric siderophores and exerts a bacteriostatic effect under iron limiting conditions. It has been observed that NGAL synthesis is induced in inflamed epithelium of the lungs and colon. Using the type II pneumocyt-derived cell line A549 as a model, we found that NGAL was up-regulated 27-fold at RNA level, and 14-fold at protein level, when co-stimulated for 24 hours with IL-17 (200ng/ml) and TNFα (20ng/ml), whereas no induction was seen with either cytokine alone compared to unstimulated cells. The expression of NGAL in A549 cells, when stimulated with IL-17 and TNFα, was dependent on de novo protein synthesis as demonstrated by inhibition of NGAL mRNA production by cycloheximide. Deletion and substitution analysis of the NGAL promoter and subsequent stimulation of transfected cells with IL-17 and TNFα showed that a functional NF-κB-binding site was essential for promoter activation whereas AP-1 and C/EBP-sites were dispensable. Quantitative RT-PCR showed a 13-fold induction of IkB-ζ mRNA in A549 cells when stimulated with IL-17 and TNFα with peak level at 1½ hours and a slow decline for up to 24 hours. Cells stimulated with IL-17 alone resulted in 8-fold mRNA induction at 1½ hours followed by a decline to a 3-fold induction level of mRNA, which lasted for 24 hours. In contrast, cells stimulated with TNFα showed a 9-fold mRNA induction at 1½ hours but then rapidly returned to back-ground level at 3 hours post-induction. These data, and previous studies of the NGAL promoter (Cowland et al. (2006), 176, 5559), demonstrates that the NGAL promoter requires binding of the transcription factor NF-κB as well as the NF-κB-binding co-factor IkB-ζ for transcriptional activation. Our data indicate that TNFα stimulates transcription of the IkB-ζ gene and NF-κB binding to the NGAL promoter whereas IL-17 stabilizes IkB-ζ mRNA and thus enables translation of this co-factor. Only when acting together the two cytokines induce the formation of an NF-κB:IκB-ζ-complex that can stimulate NGAL transcription. We have found that human b-defensin 2 (hBD2) is regulated in the same manner. The functions of NGAL and hBD2 in the innate immune response are well characterized, and the present data demonstrates that IL-17, which is synthesized only by the newly discovered TH-17 cell population, is involved in transcriptional regulation of these effector molecules. The data furthermore demonstrate that cells of the adaptive immune system is able to activate genes of the innate immune system.

Description: hCAP-18 is the only human member of the antibacterial and endotoxin-binding family of proteins known as cathelicidins. The antibacterial and endotoxin binding domains reside in the C-terminal 37 amino acids of the protein (LL-37) and this is believed to be unleashed from the neutralizing N-terminus by proteases from peroxidase positive granules. In human neutrophils, peroxidase positive and peroxidase negative granules can be subdivided into granule subsets that differ in protein content and ability to be exocytosed. To determine the localization of hCAP-18, we performed high-resolution immuno-electron microscopy and subcellular fractionation on Percoll density gradients. Biosynthesis of hCAP-18 was investigated in isolated human bone marrow cells. hCAP-18 was found to colocalize and comobilize with lactoferrin, but not with gelatinase in subcellular fractions. This was confirmed by electron microscopy. hCAP-18 is synthesized at the same stage of myeloid cell maturation as lactoferrin, and is efficiently targeted to granules. Like the peroxidase negative granule's matrix metalloproteinases, collagenase and gelatinase, hCAP-18 is also stored in unprocessed form. hCAP-18 is a major protein of specific granules where it is present in equimolar ratio with lactoferrin.

Description: Abstract 3784 Alpha-1-antitrypsin (A1AT) is an important inhibitor of the neutrophil serine proteases elastase, cathepsin G, and proteinase 3. A1AT is produced mainly by the liver and secreted to plasma. A1AT deficiency caused by the PiZZ mutation in the A1AT gene leads to accumulation of mutated A1AT in the liver which may induce liver cell necrosis and necessitate liver transplantation. In a recently performed profiling of mRNA expression during terminal neutrophil differentiation in the bone marrow, we found that A1AT mRNA increases from the promyelocyte stage and up, indicating that A1AT is a constituent of all neutrophil granules. We examined the localization and production of A1AT in healthy donor neutrophils and investigated whether the structure or function of neutrophils is affected in individuals with A1AT deficiency. RT- PCR for A1AT performed on neutrophil precursors isolated from normal human bone marrow showed that the mRNA level is highly upregulated as the cells mature in the bone marrow and even increases further as the cells enter the blood stream. Biosynthesis studies revealed that A1AT is produced by all stages of neutrophil maturation in the bone marrow and is efficiently retained in the cells as judged by pulse chase studies. Neutrophils from circulating blood also produce A1AT but this is not retained in the cells. Stimulation of neutrophils from peripheral blood with G-CSF during 24 hours resulted in a 20 fold increase in A1AT biosynthesis which was largely released to the medium. Subcellullar fractionation of blood neutrophils on a 4-layer Percoll density gradient revealed 3 forms of A1AT. A doublet band at 37 and 44 kD both with immunoreactivity against A1AT was observed in fractions corresponding to azurophil granules (cf biosynthesis of this form in promyelocytes). A band with mw of 52 kD, corresponding to the form present in blood plasma, was observed in fractions that contain NGAL, a marker of specific granules and in fractions that contain gelatinase. The 52 kD band was also observed in fractions containing albumin as expected, since secretory vesicles contain plasma proteins. The localization of A1at in neutrophil granules was further confirmed by exocytosis studies. Neutrophils were stimulated with PMA which mobilizes secretory vesicles and gelatinase granules efficiently and approximately 50% of specific granules. Only the 52 mw form of A1AT was released from cells during stimulation while none of the 37/44 double band was released. This is in agreement with localization of this double band in azurophil granules and with localization of the 52 kD form in specific granules, gelatinase granules and secretory vesicles. In addition, a high molecular weight form of A1AT was observed at 76 kD corresponding to the mw of A1AT complexed with neutrophil elastase. We isolated neutrophils from patients with the ZZ genotype of A1AT deficiency which had either been liver transplanted or lung transplanted. The neutrophils were examined by electron microscopy for detection of structural abnormalities and by exocytosis studies for detection of functional abnormalities. Electron micrographs did not reveal any abnormality in neutrophil structure and in none of the neutrophils examined (from 6 patients) did we observe abnormal granules akin to the intracellular accumulation of A1AT in liver cells from patients. We did, however, observe reduction in the total intracellular amount of A1AT in neutrophils from patients that had been lung transplanted but not in neutrophils from liver transplanted patients. This most likely reflects that secretory vesicles of neutrophils from lung transplanted will not contain A1AT as this is still severely deficient in plasma from lung transplanted patients, while liver transplanted patients will have normal levels of A1AT in plasma and hence take up normal amounts into their secretory vesicles. Release of granule proteins in response to stimulation by fMLP or PMA did not reveal any functional abnormality in neutrophils from A1AT deficient patients. Based on these studies we conclude that the A1AT deficiency does not inflict functional or structural abnormalities on neutrophils, and suggest that A1AT generated and released from neutrophils may contribute to anti-protease defense in tissues. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Olfactomedin 4 (OLFM4) is a glycoprotein predominantly expressed in myeloid cells and in gastrointestinal tissues. OLFM4 is stored in specific granules of human neutrophils where it defines a subset of neutrophils ranging from 5-40% OLFM4 positive neutrophils. OLFM4 has been proposed to inhibit cathepsin C, a cysteine protease essential for activation of serine proteases. Elevated levels of OLFM4 has been seen primary myelofibrosis (PMF) patients, these patients furthermore display a significantly elevated serum OLFM4. Methods: We developed monoclonal antibodies against OLFM4 and established an Enzyme Linked Immunosorbent Assay for OLFM4 for further investigation of OLFM4 in myeloid cells. Results: We observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 μg/mL, mean 0.028 μg/mL while approximately 10% had OLFM4 between 4 and 60 μg/mL, mean 15 μg/mL. The levels were constant over time. The level did not relate to the size of the OLFM4 positive neutrophil subset detected in peripheral blood. We studied the biosynthesis of OLFM4 in isolated bone marrow cells from an individual with high plasma OLFM4 and an individual with low plasma OLFM4. The levels of OLFM4 mRNA were comparable and the amounts of OLFM4 synthesized and retained in cells were similar between the two individuals. We next determined whether OLFM4 might be produced by bone marrow cells and released into bone marrow plasma. Corresponding levels of OLFM4 determined in bone marrow plasma and blood plasma from two persons with high levels of OLFM4 showed lower levels in bone marrow plasma than in blood plasma, arguing against bone marrow as the direct source of OLFM4 in plasma. To estimate the amount of OLFM4 generated daily during myelopoiesis, we quantitated the amount of OLFM4 in neutrophils from 3 sets of buffy coat neutrophils, each pooled from 4 healthy donors. The amount of OLFM4 was 1.2 μg/107 neutrophils. As the production of neutrophils is about 1 x 109 cells/kg body weight/day, this would indicate production of 10 mg OLFM4/day in an adult. To rule out the liver as a production site, mRNA was determined by Affymetrix gene array in liver biopsies from 42 patients evaluated for liver steatosis. OLFM4 mRNA levels were uniformly at the border of detection in all (data not shown). OLFM4 is secreted from PMA stimulated neutrophils in parallel with other specific granule proteins. When purified OLFM4 was added to medium from neutrophils induced to degranulate with PMA, the ability to detect OLFM4 was rapidly lost, indicating that OLFM4 is highly sensitive to proteolysis. Adding a cocktail of protease inhibitors to the material secreted from PMA activated neutrophils preserved OLFM4. As we find that OLFM4 is highly sensitive to proteolysis we suggest that the differences in OLFM4 levels in plasma may be related to individual differences in the susceptibility of OLFM4 to escape degradation when neutrophils decease as part of their normal life cycle. Production of 10 mg OLFM4/day would support a plasma level of 3-4 µg/ml plasma depending of the half-life of OLFM4 in plasma. This hypothesis is not easily tested, but if proven correct, might open for novel insight into the fate of neutrophils after exiting circulation, an issue that is still a matter of debate. Disclosures No relevant conflicts of interest to declare.

Description: Emergency granulopoiesis refers to the increased production of neutrophils in bone marrow and their release into circulation induced by severe infection. Several studies point to a critical role for granulocyte colony-stimulating factor (G-CSF) as the main mediator of emergency granulopoiesis. However, the consequences of G-CSF stimulation on the transcriptome of neutrophils and their precursors have not yet been elucidated in humans. Here, we investigate the changes in mRNA and miRNA expression in successive stages of neutrophil development following in vivo administration of G-CSF in humans, mimicking emergency granulopoiesis. Blood samples were collected from healthy individuals after five days of G-CSF administration. Neutrophil precursors were sorted into discrete stages of maturation by flow cytometry and extracted RNA was subjected to microarray analysis. mRNA levels were compared to previously published expression levels in corresponding populations of neutrophil precursors isolated from bone marrow of untreated, healthy individuals. miRNA expression was investigated in the most mature cell population to determine G-CSF-induced changes in circulating neutrophils. G-CSF substantially affected mRNA and miRNA expression patterns, demonstrating significant impact on neutrophil development and function. 1110 mRNAs were differentially expressed more than 2-fold with G-CSF while the treatment induced changes in the levels of 73 miRNAs in the mature population. In addition, G-CSF treatment reduced the levels of four out of five measured granule proteins in mature neutrophils including hCAP-18, which was completely deficient in neutrophils from G-CSF-treated donors. Cell cycle analysis pointed towards an induced proliferative capacity of myelocytes. These results indicate that multiple biological processes are altered in order to satisfy the increased demand for neutrophils during G-CSF-induced emergency granulopoiesis. Disclosures No relevant conflicts of interest to declare.

Description: To characterize the transcriptional program that governs terminal granulocytic differentiation in vivo, we performed comprehensive microarray analyses of human bone marrow (BM) populations highly enriched in promyelocytes (PMs), myelocytes/metamyelocytes (MYs), and neutrophils (bm-PMNs). These analyses identified 11,310 genes involved in differentiation, of which 6,700 were differentially regulated including granule proteins and surface receptors previously not identified in neutrophils. Functional clustering demonstrated that differentially expressed genes were assigened to all of the major gene categories including apoptosis, cell cycle, chaperones, enzymes, immunity proteins, kinases, motor proteins, signal transducers, structural proteins, and transcription factors. In an attempt to define the developmental distance between PM, MY, and bm-PMN populations, we assessed the Pearsons correlation coefficient (γ) by correlating the transcriptomes (=all 44.760 probe sets, Affymetrix) of BM populations. The correlation coefficients among replicates of BM populations were in the range of 0.97–0.99. On the other hand the correlation coefficients between BM populations were 0.81 (γPM - MY), 0.79 (γMY - bm-PMN), and 0.52 (γPM - bm-PMN), and thus, provided a useful quantitative measure reflecting the hierarchical relationship between the three BM populations. Additional correlation of bm-PMN and peripheral blood-PMN transcriptomes revealed a high similarity of both populations (γbm-PMN - pb-PMN=0.95) indicating a termination of granulocytic differentiation in the BM microenvironment during steady state hematopoesis. Differentiation of PMs towards MYs was accompanied by a marked decline of proliferative and general cellular activity as defined by downregulation of E2F target genes, cyclin dependent kinases 2/4/6, 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 upregulated in bm-PMNs. The upregulation of ligand-receptor pairs, which are defined inducers as well as target genes of NF-kB, suggests a constitutive activation of NF-kB in bm-PMNs by autocrine loops. Overall, these results define a granulocytic differentiation model governed by a highly coordinated fail-safe program, which promotes completion of differentiation before cells gain responsiveness towards activating stimuli that accompany infections.

Description: Abstract 2136 Formation of polymorphonuclear neutrophils (PMN) is a tightly regulated process where the myeloid progenitor cells, myeloblasts (MBs), divide and mature in the bone marrow, along a well defined path. The cells pass through six well defined stages in differentiation ending up with the release of mature PMNs to peripheral blood (granulopoiesis). Expression of essential transcription factors such as RUNX1, C/EBP-a, and C/EBP-e during granulopoiesis has been shown to have great importance for correct neutrophil development. microRNAs (miRNAs) could be important players in the fine-tuning of transcription factor expression due to their ability to regulate protein synthesis. The function of neutrophils is to detect and destroy invading microorganisms. This involves activation of the PMNs in the blood stream causing a release of secretory vesicles and up-regulation of extracellular adhesion molecules followed by migration in the tissue towards the focus of inflammation. Expression of miRNAs might also be regulated during activation and diapedesis of the neutrophils in order to adapt the neutrophil to its new environment and function. A regulatory role for miRNAs has been demonstrated for several biological processes, such as proliferation, differentiation, inflammation and cancer, and dysregulation of miRNA expression has been shown to contribute to disease development. The purpose of this study was to determine the miRNA expression profiles during normal human granulopoiesis starting with the first identifiable granulocytic precursor cell (MB) and ending with activated neutrophils that have migrated into the tissue using an Affymetrix 2,0 miRNA microarray platform. We isolated four populations of cells: Myeloblasts (MB) and promyelocytes (PM), myelocytes (MC) and metamyelocytes (MM), and band cells (BC) and segmented cells (SC) from the bone marrow and PMNs from peripheral blood from three different donors. We found 135 differentially expressed miRNAs in granulopoiesis, which could be divided into six clusters according to their expression pattern. 87% of the 135 miRNAs were differentially regulated between the MB/PM (dividing cells) and the MC/MM stages (cessation of cell proliferation and initiation of terminal differentiation) and could imply a need for miRNA-mediated regulation of the many proteins involved in regulating this process. Interestingly, we also found two distinct clusters of miRNAs that were either up- or down-regulated only in the MC/MM population, indicating the importance of a specific temporary regulation of some proteins during neutrophil development. To determine miRNA expression profiles in activated granulocytes, we examined PMNs and activated neutrophils from skin window (i.e. PMNs migrated to a site of inflammation). We found seven differentially expressed miRNAs, - all of them up regulated in the activated neutrophils. Using microRNA target-prediction software, we found that miRNAs 155, 146a and 130a, all of which are strongly up-regulated in the MB/PM stage, have several targets in the IL1-receptor signalling cascade, indicating the importance of miRNA of dampening an innate immune response in immature neutrophil precursors. miR-146a, 155 and 130a also have predicted targets in either the TGF-βI or the TGF-βII receptor which inhibits proliferation when binding to TGF-β. This finding supports the proliferating profile for the MB/PM cells, and the shift towards cell cycle arrest when the cells differentiate to the next stage, where expression of these three miRNAs is low. miRNA-34c-3p is highly expressed only in the MC/MM stage and has verified targets in many different mRNAs involved in the regulation of cell cycle arrest. All the miRNAs that were up-regulated in the activated neutrophils have several predicted targets in the IL1R pathway, and some of them (miR-212, −132 and −297) have previously been shown to be important in regulating the inflammatory response. The study indicates that several different miRNAs have important roles in the regulation of normal granulopoiesis, and that miRNAs also might be part of a possible negative feed back loop in the inflammatory response in activated neutrophils. Grant acknowledgments: The Danish Cancer Society, Lundbeck foundation, Danish Medical Research Council, Brøchner Mortensen foundation Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Saliva induces bactericidal and DNase resistant NETs in the oral cavity via sialyl LewisX- L-selectin signaling. Disordered homeostasis in the oral cavity may lead to deficient saliva-mediated NETosis.

Description: The mechanism by which proteins are targeted to neutrophil granules is largely unknown. The intracellular proteoglycan serglycin has been shown to have important functions related to storage of proteins in several types of granules. The possible role of serglycin in the localization of the α-defensin, human neutrophil peptide 1 (HNP-1), a major azurophil granule protein in human neutrophils, was investigated. Murine myeloid cells, stably transfected to express HNP-1, were capable of processing HNP-1, and HNP-1 was found to associate with serglycin in murine and human myeloid cell lines as well as in human bone marow cells. A transgenic mouse expressing HNP-1 in the myeloid compartment was crossed with mice deficient in serglycin or neutrophil elastase to investigate HNP-1 sorting and processing. Neither deficiency affected processing of HNP-1, but the ability to retain fully processed HNP-1 intracellularly was reduced in mice that lack serglycin. Human granulocyte precursors transfected with siRNA against serglycin displayed similar reduced capability to retain fully processed HNP-1, demonstrating a role of serglycin in retaining mature HNP-1 intracellularly, thus preventing potential toxic effects of extracellular HNP-1.

Description: Smad4 is important in the TGF-β pathway and required for transcriptional activation and inhibition of cell growth after TGF-β1 stimulation. We demonstrate that miR-130a is differentially expressed during granulopoiesis and targets Smad4 mRNA. The transcript for Smad4 is present throughout neutrophil maturation, but Smad4 protein is undetectable in the most immature cells, where miR-130a is highly expressed. Two miR-130a binding sites were identified in the 3′-untranslated region of the Smad4 mRNA. Overexpression of miR-130a in HEK293, A549, and 32Dcl3 cells repressed synthesis of Smad4 protein without affecting Smad4 mRNA level. Repression of Smad4 synthesis in a granulocytic cell line by miR-130a reduced its sensitivity to TGF-β1–induced growth inhibition. This effect was reversed by inhibiting the activity of miR-130a with an antisense probe or by expressing a Smad4 mRNA lacking miR-130a binding sites. High endogenous miR-130a and Smad4 mRNA levels and low expression of Smad4 protein were found in the t(8;21)(q22;q22) acute myelogenous leukemia–derived cell line Kasumi-1. When miR-130a was inhibited by an antisense RNA, the amount of Smad4 protein increased in Kasumi-1 cells and rendered it susceptible for TGF-β1–mediated cell growth inhibition. Our data indicate that miR-130a is involved in cell cycle regulation of granulocytic cells through engagement of Smad4 in the TGF-β pathway.