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A Drug Response Prediction (DRP) score was developed based on gene expression profiling (GEP) from cell lines and tumor samples. Twenty percent of high-risk patients by GEP70 treated in Total Therapy 2 and 3A have a progression-free survival (PFS) of more than 10years. We used available GEP data from high-risk patients by GEP70 at diagnosis from Total Therapy 2 and 3A to predict the response by the DRP score of drugs used in the treatment of myeloma patients. The DRP score stratified patients further. High-risk myeloma with a predicted sensitivity to melphalan by the DRP score had a prolonged PFS, HR=2.4 (1.2-4.9, P=0.014) and those with predicted sensitivity to bortezomib had a HR 5.7 (1.2-27, P=0.027). In case of predicted sensitivity to bortezomib, a better response to treatment was found (P=0.022). This method may provide us with a tool for identifying candidates for effective personalized medicine and spare potential non-responders from suffering toxicity.

To the editor: Lipocalin-2 is a eukaryotic siderophore-binding protein that prevents the growth and spread of microorganisms that require siderophore-mediated uptake of soluble iron.[1][1] Consequently, lipocalin-2 knockout mice ( Lcn2 −/− mice) are more susceptible to infection with the

Recent studies show that mantle cell lymphoma (MCL) express aberrant microRNA (miRNA) profiles; however, the clinical effect of miRNA expression has not previously been examined and validated in large prospective homogenously treated cohorts. We performed genome-wide miRNA microarray profiling of 74 diagnostic MCL samples from the Nordic MCL2 trial (screening cohort). Prognostic miRNAs were validated in diagnostic MCL samples from 94 patients of the independent Nordic MCL3 trial (validation cohort). Three miRNAs (miR-18b, miR-92a, and miR-378d) were significantly differentially expressed in patients who died of MCL in both cohorts. MiR-18b was superior to miR-92a and miR-378d in predicting high risk. Thus, we generated a new biological MCL International Prognostic Index (MIPI-B)-miR prognosticator, combining expression levels of miR-18b with MIPI-B data. Compared to the MIPI-B, this prognosticator improved identification of high-risk patients with regard to cause-specific, overall, and progression-free survival. Transfection of 2 MCL cell lines with miR-18b decreased their proliferation rate without inducing apoptosis, suggesting that miR-18b may render MCL cells resistant to chemotherapy by decelerating cell proliferation. We conclude that overexpression of miR-18b identifies patients with poor prognosis in 2 large prospective MCL cohorts and adds prognostic information to the MIPI-B. MiR-18b may reduce the proliferation rate of MCL cells as a mechanism of chemoresistance.

Lipocalin-2 (LCN2) was originally isolated from human neutrophils and termed neutrophil gelatinase-associated lipocalin (NGAL). However, the functions of LCN2 and the cell types that are primarily responsible for LCN2 production remain unclear. To address these issues, hepatocyte-specific Lcn2 knockout (Lcn2(Hep-/-)) mice were generated and subjected to bacterial infection (with Klesbsiella pneumoniae or Escherichia coli) or partial hepatectomy (PHx). Studies of Lcn2(Hep-/-) mice revealed that hepatocytes contributed to 25% of the low basal serum level of LCN2 protein (∼ 62 ng/mL) but were responsible for more than 90% of the highly elevated serum LCN2 protein level (∼ 6,000 ng/mL) postinfection and more than 60% post-PHx (∼ 700 ng/mL). Interestingly, both Lcn2(Hep-/-) and global Lcn2 knockout (Lcn2(-/-)) mice demonstrated comparable increases in susceptibility to infection with K. pneumoniae or E. coli. These mice also had increased enteric bacterial translocation from the gut to the mesenteric lymph nodes and exhibited reduced liver regeneration after PHx. Treatment with interleukin (IL)-6 stimulated hepatocytes to produce LCN2 in vitro and in vivo. Hepatocyte-specific ablation of the IL-6 receptor or Stat3, a major downstream effector of IL-6, markedly abrogated LCN2 elevation in vivo. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that STAT3 was recruited to the promoter region of the Lcn2 gene upon STAT3 activation by IL-6. Conclusion Hepatocytes are the major cell type responsible for LCN2 production after bacterial infection or PHx, and this response is dependent on IL-6 activation of the STAT3 signaling pathway. Thus, hepatocyte-derived LCN2 plays an important role in inhibiting bacterial infection and promoting liver regeneration.

Introduction The label-free dynamic mass redistribution-based assay (DMR) is a powerful method for studying signalling pathways of G protein-coupled receptors (GPCRs). Herein we present the label-free DMR assay as a robust readout for pharmacological characterization of formyl peptide receptors (FPRs) in human neutrophils. Methods Neutrophils were isolated from fresh human blood and their responses to FPR1 and FPR2 agonists, i.e. compound 43, fMLF and WKYMVm were measured in a label-free DMR assay using Epic Benchtop System from Corning®. Obtained DMR traces were used to calculate agonist potencies. Results The potencies (pEC 50 ) of fMLF, WKYMVm and compound 43, determined on human neutrophils using the label-free DMR assay were 8.63, 7.76 and 5.92, respectively. The DMR response to fMLF, but not WKYMVm and compound 43 could be blocked by the FPR1-specific antagonist cyclosporin H. Discussion We conclude that the DMR assay can be used, and complements more traditional methods, to study the signalling and pharmacology of endogenous FPR receptors in human neutrophils.

A memorial for Niels Borregaard, M.D., Ph.D.

t(8;21) acute myeloid leukemia (AML) is characterized by a translocation between chromosomes 8 and 21 and formation of a distinctive RUNX1-RUNX1T1 fusion transcript. This translocation places RUNX1T1 under control of the RUNX1 promoter leading to a pronounced upregulation of RUNX1T1 transcripts in t(8;21) AML, compared to normal hematopoietic cells. We investigated the role of highly-upregulated RUNX1T1 under the hypothesis that it acts as competing endogenous RNA (ceRNA) titrating microRNAs (miRNAs) away from their target transcripts and thus contributes to AML formation. Using publicly available t(8;21) AML RNA-Seq and miRNA-Seq data available from The Cancer Genome Atlas (TCGA) project, we obtained a network consisting of 605 genes that may act as ceRNAs competing for miRNAs with the suggested RUNX1T1 miRNA sponge. Among the 605 ceRNA candidates, 121 have previously been implied in cancer development. Players in the integrin, cadherin, and Wnt signaling pathways affected by the RUNX1T1 sponge were overrepresented. Finally, among a set of 21 high interest RUNX1T1 ceRNAs we found multiple genes that have previously been linked to AML formation. In conclusion, our study offers a novel look at the role of the RUNX1-RUNX1T1 fusion transcript in t(8;21) AML beyond previously investigated genetic and epigenetic aberrations.

Granules are essential for the ability of neutrophils to fulfill their role in innate immunity. Granule membranes contain proteins that react to environmental cues directing neutrophils to sites of infection and initiate generation of bactericidal oxygen species. Granules are densely packed with proteins that contribute to microbial killing when liberated to the phagosome or extracellularly. Granules are, however, highly heterogeneous and are traditionally subdivided into azurophil granules, specific granules, and gelatinase granules in addition to secretory vesicles. This review will address issues pertinent to formation of granules, which is a process intimately connected to maturation of neutrophils from their precursors in the bone marrow. We further discuss possible mechanisms by which decisions are made regarding sorting of proteins to constitutive secretion or storage in granules and how degranulation of granule subsets is regulated.

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 (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.

Neutrophil gelatinase-associated lipocalin (NGAL) is a siderophore-binding antimicrobial protein that is up-regulated in epithelial tissues during inflammation. We demonstrated previously that the gene encoding NGAL (LCN2) is strongly up-regulated by interleukin (IL)-1beta in an NF-kappaB-dependent manner but not by tumor necrosis factor (TNF)-alpha, another potent activator of NF-kappaB. This is due to an IL-1beta-specific synthesis of the NF-kappaB-binding co-factor IkappaB-zeta, which is essential for NGAL induction. We demonstrate here that NGAL is strongly induced by stimulation with TNF-alpha in the presence of IL-17, a pro-inflammatory cytokine produced by the newly discovered subset of CD4(+) T helper cells, T(H)-17. In contrast to the murine NGAL orthologue, 24p3/lipocalin 2, we found no requirement for C/EBP-beta or C/EBP-delta for NGAL induction by IL-17 and TNF-alpha as neither small interfering RNAs against the two C/EBP mRNAs nor mutation of the C/EBP sites in the LCN2 promoter abolished IL-17- and TNF-alpha-induced up-regulation of NGAL. NGAL induction is governed solely by NF-kappaB and its co-factor IkappaB-zeta. This was demonstrated by a pronounced reduction in the amount of NGAL mRNA and NGAL protein synthesized in cells treated with small interfering RNA against IkappaB-zeta and a total lack of activation of an LCN2 promoter construct with a mutated NF-kappaB site. As IL-17 stimulation stabilizes the IkappaB-zeta transcript, we propose a model where TNF-alpha induces activation and binding of NF-kappaB to the promoters of both NFKBIZ and LCN2 genes but induce only transcription of IkappaB-zeta. Co-stimulation with IL-17 leads to accumulation of IkappaB-zeta mRNA and IkappaB-zeta protein, which can bind to NF-kappaB on the LCN2 promoter and thus induce NGAL expression.

Ficolins constitute a family of proteins whose biological role has been an enigma for many years. Over the past few years it has become evident that ficolins are part of the innate immune system and function as recognition molecules in the complement system. The 3 human ficolins, ficolin-1 (M-ficolin), ficolin-2 (L-ficolin) and ficolin-3 (H-ficolin or Hakata antigen) are encoded by the FCN1, FCN2 and FCN3 genes, respectively. Phylogenetic studies suggest that ficolins are of ancient origin. Ficolin-3 seems to be the most ancient molecule, from a phylogenetic perspective. Searches in databases and phylogenetic tree analysis demonstrate that the ficolin precursor has gone through an expansion involving independent duplication events in the different branches of the evolutionary tree. Of particular interest is the prediction that ficolin-1 appears to be present as an ortholog molecule. All human FCN genes are polymorphic. The FCN2 gene encoding ficolin-2, contains polymorphisms that affect ligand binding, while differences in the serum levels are associated with promoter polymorphisms. Recently, a frame-shift variation in the FCN3 gene was described, leading to ficolin-3 deficiency and defective complement activation. This FCN3 variation was also shown to be associated with immunodeficiency. This survey summarizes the current phylogenetic and inter-individual molecular understanding of the FCN genes.

Storage and release of proteins from granules forms the basis of cellular functions as diverse as cell mediated cytotoxicity, neuronal communication, activation of muscle fibres, and release of hormones or digestive enzymes from endocrine and exocrine glands, such as the pancreas. Serglycin is the major intracellular proteoglycan of haematopoietic cells. Serglycin is important for localization of proteins in granules of different haematopoietic cell types. Previous reports have indicated a role for serglycin in granule formation and localization of zymogens in granules of the exocrine pancreas in rat. We here present data showing that serglycin is not present at the protein level in human or murine pancreas. Furthermore, the amount and localization of three exocrine pancreas zymogens (amylase, trypsinogen, and carboxypeptidase A) is not affected by the absence of serglycin in a serglycin knock-out mouse model.

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.

Objective Sjogren's syndrome (SS), an autoimmune disease of exocrine glands, typically starts at the time of adrenopause. We undertook this study to test the hypothesis that SS is characterized by an insufficient androgen effect at the target tissue level. Methods We searched for androgen response elements (AREs) in the cysteine-rich secretory protein 3 (crisp-3) gene. Dehydroepiandrosterone (DHEA) responsiveness was experimentally studied using quantitative reverse transcriptase–polymerase chain reaction and immunofluorescence staining of human submandibular gland–derived acinar cells and labial salivary gland explants with or without DHEA. Finally, glandular and salivary CRISP-3 in healthy controls and SS patients was analyzed using immunohistochemistry, in situ hybridization, and enzyme-linked immunosorbent assay. Serum DHEA sulfate (DHEAS) and salivary DHEA levels were measured using a radioimmunometric method. Results Literature analysis and a search for AREs in gene banks suggested androgen dependency of human CRISP-3, and this was verified by studies of human submandibular gland acinar cells cultured with or without DHEA, in which DHEA increased CRISP-3 messenger RNA (mRNA) levels (P = 0.018). This finding was confirmed by the results of DHEA stimulation of labial salivary gland explants. Glandular CRISP-3 mRNA and protein labeling was weak and diffuse, coupled with low secretion in saliva (mean ± SEM 21.1 ± 2.7 μg CRISP-3/15 minutes in SS patients versus 97.6 ± 12.0 μg CRISP-3/15 minutes in healthy controls; P < 0.0001). Compared with healthy controls, SS patients had low serum levels of DHEAS (P = 0.008) and also low salivary levels of DHEA (mean ± SEM 224 ± 33 pmoles versus 419 ± 98 pmoles; P = 0.005). Conclusion CRISP-3 pathology was seen in acini remote from lymphocyte foci and is apparently not secondary to local inflammation, but may represent some systemic effect in SS. Indeed, androgen deprivation in the salivary glands of SS patients is evidenced both by low salivary levels of DHEA and by low levels of DHEA-regulated CRISP-3. This may explain some of the characteristic features of SS.

Background MicroRNAs (miRNAs) are important for the development and function of neutrophils. miR-130a is highly expressed during early neutrophil development and regulates target proteins important for this process. miRNA targets are often identified by validating putative targets found by in silico prediction algorithms one at a time. However, one miRNA can have many different targets, which may vary depending on the context. Here, we investigated the effect of miR-130a on the proteome of a murine and a human myeloid cell line. Results Using pulsed stable isotope labelling of amino acids in cell culture and mass spectrometry for protein identification and quantitation, we found 44 and 34 proteins that were significantly regulated following inhibition of miR-130a in a miR-130a-overexpressing 32Dcl3 clone and Kasumi-1 cells, respectively. The level of miR-130a inhibition correlated with the impact on protein levels. We used RAIN, a novel database for miRNA–protein and protein–protein interactions, to identify putative miR-130a targets. In the 32Dcl3 clone, putative targets were more up-regulated than the remaining quantified proteins following miR-130a inhibition, and three significantly derepressed proteins (NFYC, ISOC1, and CAT) are putative miR-130a targets with good RAIN scores. We also created a network including inferred, putative neutrophil miR-130a targets and identified the transcription factors Myb and CBF-β as putative miR-130a targets, which may regulate the primary granule proteins MPO and PRTN3 and other proteins differentially expressed following miR-130a inhibition in the 32Dcl3 clone. Conclusion We have experimentally identified miR-130a-regulated proteins within the neutrophil proteome. Linking these to putative miR-130a targets, we provide an association network of potential direct and indirect miR-130a targets that expands our knowledge on the role of miR-130a in neutrophil development and is a valuable platform for further experimental studies. Electronic supplementary material The online version of this article (doi:10.1186/s12865-015-0134-8) contains supplementary material, which is available to authorized users.

The α-defensins human neutrophil peptides (HNPs) are the predominant antimicrobial peptides of neutrophil granules. They are synthesized in promyelocytes and myelocytes as proHNPs, but only processed and stored as mature HNPs in promyelocytes. Despite decades of search, the mechanisms underlying the posttranslational processing of neutrophil defensins remain unidentified. Thus, neither the enzyme processing proHNPs nor the localization of processing has been identified. It has long been hypothesized that proHNPs are processed by the serine proteases highly expressed in promyelocytes: Neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3), all of which have shown in vitrocapability to process recombinant proHNP into HNP. We investigated, whether serine proteases are in fact responsible for proHNP processing in human bone marrow cells and in human and murine myeloid cell lines. Subcellular fractionation of the human promyelocytic cell line PLB-985 demonstrated proHNP processing to commence in fractions containing endoplasmic reticulum. Processing of 35S-proHNP was insensitive to serine protease inhibitors. Simultaneous knockdown of NE, CG, and PR3 did not decrease proHNP processing by primary human neutrophil precursors. Furthermore, introduction of NE, CG, and PR3 into murine promyelocytic cells did not increase processing capability. Finally, two patients suffering from Papillon–Lefèvre syndrome, who lack the ability to activate neutrophil serine proteases, demonstrated normal levels of fully processed HNP in peripheral neutrophils. Contradicting earlier assumptions, our study found serine proteases dispensable for processing of proHNPs in vivo. This calls for study of other protease classes in the search for the proHNP processing protease(s). Disclosures No relevant conflicts of interest to declare.

Bactericidal/permeability-increasing protein (BPI) neutralizes the proinflammatory effects of lipopolysaccharide and is of potential clinical use in the treatment of fulminant Gram-negative infections. BPI is a cationic protein with antibacterial activity stored in azurophil (primary) granules of neutrophil granulocytes. However, the absence of BPI in patients with specific granule deficiency indicates a transcriptional control of BPI, which is distinct from that of other azurophil granule proteins. Accordingly, we demonstrate in vivo that the BPI mRNA level peaks, together with mRNA for specific granule proteins, during the myelocytic and metamyelocytic stage of granulocytic maturation. The human promyelocytic cell line NB4 expresses several azurophil granule proteins, but expression of BPI is undetectable. We show that treatment of NB4 cells with all-trans retinoic acid (ATRA) induces BPI expression at mRNA and at protein level. The induction is dependent on de novo protein synthesis, as judged by sensitivity to cycloheximide. Previous investigations have indicated a potential role of CCAAT/enhancer-binding protein (C/EBP) transcription factors in the regulation of BPI expression. Here, we show that induction of NB4 cells with ATRA correlates to direct binding of C/EBPβ and C/EBPε to the proximal BPI promoter, as determined by electrophoretic mobility shift analysis and chromatin immunoprecipitation. The dependency on C/EBPβ and C/EBPε provides an explanation for delayed BPI mRNA expression, as compared with mRNA of other azurophil granule proteins.

The pathogenesis of chronic lymphocytic leukemia (CLL) is unknown but may involve aberrant activation of signaling pathways. Somatic hypermutations in rearranged immunoglobulin heavy-chain (IgVH) genes allow a division of CLL patients into 2 categories: mutated IgVH genes are associated with an indolent disease, whereas unmutated IgVH genes define an aggressive form. Using differential display to compare gene expression in CLL cells with and without IgVH hypermutations, we identified a novel gene, CLL up-regulated gene 1 (CLLU1), that was highly up-regulated in CLL cells without IgVH hypermutations. CLLU1 mapped to chromosome 12q22, within a cluster of genes that are active in germinal center B cells. However, appreciable levels of CLLU1 were detectable only in CLL cells and not in a panel of normal tissue extracts or in any other tested hematologic malignancy. High expression of CLLU1 in CLL samples occurred irrespective of trisomy 12 or large chromosomal rearrangements. CLLU1 encodes 6 mRNAs with no sequence homology to any known gene, and most transcripts appear to be noncoding. Two transcripts, however, potentially encode a peptide with remarkable structural similarity to human interleukin 4. These data, in particular the unique and restricted expression pattern, suggest that CLLU1 is the first disease-specific gene identified in CLL.

Defensins are potent antimicrobial and proinflammatory peptides. The human neutrophil defensins human neutrophil peptide (HNP)-1–3 are synthesized as 94 amino acide (aa) preproHNPs, which are converted to 75 aa proHNPs by cotranslational removal of a 19 aa endoplasmic reticulum signal peptide. At the promyelocytic stage of myelopoiesis, proHNPs are further proteolytically modified and accumulate in azurophil granules as 29–30 aa HNPs. In contrast, proHNPs produced by more mature myeloid cells are not subjected to proteolytic cleavage and undergo a high degree of constitutive exocytosis. The proHNPs are devoid of antimicrobial potential, and the significance of their secretion is unknown. To investigate whether mature neutrophils contain proHNPs, we developed antibodies against proHNP-1 by DNA immunization of rabbits. In addition, antibodies against the 45 aa proHNP pro-piece were raised by conventional immunization procedures. These antibodies allowed detection of proHNPs in homogenates of peripheral blood neutrophils. The proHNPs were isolated by affinity chromatography, and their identity was confirmed by mass spectrometry and N-terminal aa sequence analysis. Finally, the neutrophil proHNPs were identified as novel matrix proteins of specific granules by subcellular fractionation experiments, release studies, and immunoelectron microscopy. Thus, human neutrophils not only store large amounts of mature defensins in azurophil granules but also contain a more easily mobilized reservoir of unprocessed prodefensins in specific granules.

Serglycin is a major proteoglycan of hematopoietic cells. It is thought to play a role in the packaging of granule proteins in human neu- trophil granulocytes. The presence of serglycin in myeloid cells has been demonstrated only at the transcriptional level. We generated a polyclonal antibody against recombinant human serglycin. Here, we show the localization of serglycin in hu- mans during neutrophil differentiation. Immuno- cytochemistry revealed serglycin immunoreactiv- ity in the Golgi area of promyelocytes (PM) and myelocytes (MC), as well as in a few band cells and mature neutrophil granulocytes. Granular staining was detected near the Golgi apparatus in some of the PM, and the major part of the cytoplasm was negative. Immunoelectron microscopy showed ser- glycin immunoreactivity located to the Golgi appa- ratus and a few immature granules of PM and MC. The decreasing level of serglycin protein during myeloid differentiation coincided with a decrease of mRNA expression, as evaluated by Northern blotting. Subcellular fractions of neutrophil gran- ulocytes were obtained. Serglycin immunoreactiv- ity was detected in the fraction containing Golgi apparatus, plasma membrane, and secretory vesi- cles by Western blotting and enzyme-linked immu- nosorbent assay. Serglycin was not detected in sub- cellular fractions containing primary, secondary, or tertiary granules. Together, these findings indi- cate that serglycin is located to the Golgi apparatus and a few immature granules during neutrophil differentiation. This is consistent with a function for serglycin in formation of granules in neutrophil granulocytes. Our findings contrast the view that native serglycin is present in mature granules and plays a role in packaging and regulating the activity of proteolytic enzymes there. J. Leukoc. Biol. 76: 406-415; 2004.

The in vivo expression profiles of cell-cycle proteins regulating G1-to-S-phase transition were determined in three neutrophil precursor populations from human bone marrow: myeloblasts (MBs) and promyelocytes (PMs); myelocytes (MCs) and metamyelocytes (MMs); and band cells (BCs) and segmented neutrophil cells (SCs) and in mature polymorphonuclear neutrophils (PMNs) from peripheral blood. Complete cell-cycle arrest was observed in BCs/SCs and PMNs. Cyclins D1, D2, and D3 were found to be down-regulated during granulopoiesis, whereas a slight increase of cyclin E was seen. In contrast, cyclin-dependent kinase (CDK)2, -4, and -6 were down-regulated from the MC/MM stages and onward. The transcript levels of CDK2, -4, and -6 were concurrently down-regulated. As the only CDK inhibitor, p27kip1 protein and mRNA expression were up-regulated in MCs/MMs and reached peak levels in PMNs. Protein expression of retinoblastoma protein and the related pocket proteins p107 and p130 was down-regulated from the MC/MM stages and onward. This is the first report to describe expression levels of cell-cycle proteins during granulopoiesis in vivo, and it strongly contrasts the observations made in cell-culture systems in vitro.

Synthesis of the antimicrobial protein neutrophil gelatinase-associated lipocalin (NGAL) increases dramatically in bronchial epithelial cells and alveolear type II pneumocytes during lung inflammation. IL-1β induces a >10-fold up-regulation of NGAL expression in the type II pneumocyte-derived cell line A549 cells, whereas TNF-α, IL-6, and LPS had no effect. Similar IL-1β selectivity was demonstrated in primary bronchial epithelial cells and epidermal keratinocytes and for an NGAL promoter fragment transfected into A549 cells. By deletion and substitution analysis of the NGAL promoter, a 40-bp region containing an NF-κB consensus site was found to control the IL-1β-specific up-regulation. Involvement of the NF-κB site was demonstrated by site-directed mutagenesis, by transfection with a dominant-negative inhibitor of the NF-κB pathway, and by EMSA. TNF-α activation of NF-κB, in contrast, did not increase NGAL synthesis, even though induced binding of NF-κB to the NGAL promoter was observed in vitro. IL-1β specificity was not contained within the NF-κB site of the NGAL promoter, as determined by exchanging the NGAL promoter′s NF-κB-binding sequence with that of the IL-8 promoter or with the NF-κB consensus sequence and by testing the NF-κB-binding sequence of the NGAL promoter against the heterologous SV40 promoter. Selectivity for the IL-1 pathway was substantiated by demonstrating that NGAL promoter activity could be induced by LPS stimulation of A549 cells transiently expressing Toll-like receptor 4, which use the same intracellular signaling pathway as the IL-1R. Together, this demonstrates a selective up-regulation of NGAL by the IL-1 pathway.

In vivo distribution of myeloid transcription factors during granulopoiesis was investigated by Northern and Western blotting in 3 neutrophil precursor populations from human bone marrow: immature (myeloblasts [MBs] and promyelocytes [PMs]); intermediate mature (myelocytes [MCs] and metamyelocytes [MMs]); and mature neutrophil cells (band cells [BCs] and segmented neutrophil cells [SCs]). Nonneutrophil cells were removed with magnetic-bead–coupled antibodies against CD2, CD3, CD14, CD19, CD56, CD61, glycophorin-A, and CD49d (BCs/SCs) before RNA and protein extraction. Polymorphonuclear neutrophils (PMNs) from peripheral blood depleted with anti-CD49d antibodies were also included. Expression of acute myeloid leukemia 1b (AML-1b), c-myb, GATA-1, and CCAAT/enhancer binding protein γ (C/EBP-γ) was seen primarily in MBs/PMs, and little expression was found in more mature cells. The level of C/EBP-α was constant in the bone marrow–derived cells and decreased in PMNs. C/EBP-ϵ was found primarily in MCs/MMs and was almost absent in more mature cells. Expression of C/EBP-β, C/EBP-δ, and C/EBP-ζ was observed from the MC/MM stage onward, with peak levels in the most mature cells. The amount of PU.1 increased throughout maturation whereas the level of Elf-1 reached a nadir in MCs/MMs The PU.1 coactivator c-jun and c-jun's dimerization partner c-fos were both detectable in MCs/MMs and increased in amount with maturity. CCAAT displacement protein (CDP) was found at comparable levels at all stages of differentiation. This demonstrates a highly individualized expression of the transcription factors, which can form the basis for the heterogeneous expression of granule proteins during granulopoiesis and cell cycle arrest in metamyelocytes.

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa protein initially isolated from the specific granules of human neutrophils. It is a member of the highly heterogeneous lipocalin protein family, which shares a common tertiary structure. Its synthesis is induced in gastrointestinal epithelium in association with inflammation and malignancy. To gain insight into its potential role in other epithelia we have investigated the expression of NGAL in human skin embryonic development, in normal adult skin, and in skin associated with inflammation and neoplastic transformation. In the present study we report that the embryonic expression of NGAL appears to be regulated in a spatio-temporal pattern. It was induced in the interfollicular epidermis at 20-24 weeks of gestational age but thereafter progressively receded towards the hair follicles. In normal adult skin, NGAL was detected solely in association with hair follicles. However, strong induction of NGAL in the epidermis was seen in a variety of skin disorders characterized by dysregulated epithelial differentiation such as psoriasis, pityriasis rubra and squamous cell carcinoma. In these tissues production of NGAL was confined to spatially distinct subpopulations of keratinocytes underlying areas of parakeratosis, whereas skin samples lacking parakeratotic epithelium such as lichen ruber planus, acute contact eczema and basal cell carcinoma were negative for NGAL. Consistent with being a marker for disturbed terminal differentiation, NGAL immunoreactivity showed an inverse pattern when compared with that of the differentiation marker filaggrin. The biologic functions of NGAL in epithelia are not fully known, although an immunomodulatory role in host defense has been proposed. In addition, the transient interfollicular NGAL expression during skin embryogenesis along with the induction of NGAL in adult parakeratotic epidermis suggests it play a role in epithelial differentiation pathways.

Specific granule protein of 28 kDa (SGP28), also termed cysteine-rich secretory protein 3 (CRISP-3), is a glycoprotein that belongs to a family of cysteine-rich secretory proteins (CRISPs). SGP28 was originally discovered in human neutrophils, but transcripts are widely distributed in exocrine glands (salivary glands, pancreas, and prostate) and also found at lower levels in epididymis, ovary, thymus, and colon. The function of SGP28/CRISP-3 is not yet known. Similarities to pathogenesis-related proteins in plants and the expression in neutrophils and exocrine glands suggest that SGP28/CRISP-3 may play a role in innate host defense. We describe here the production of a recombinant, C-terminally truncated form of CRISP-3 (rCRISP-3Delta) and the generation of polyclonal antibodies against rCRISP-3Delta that are useful in immunoblotting and immunocytochemistry. We present a specific, accurate, and reproducible enzyme-linked immunosorbant assay (ELISA) for the measurement of CRISP-3 with a detection limit of 2 ng/ml. We further demonstrate the presence of CRISP-3 protein in human plasma (6.3 microg/ml), saliva (21.8 microg/ml), seminal plasma (11.2 microg/ml), and sweat (0.15 microg/ml), and describe the coexistence of two different molecular weight forms of CRISP-3, representing an N-glycosylated and a non-glycosylated form of the mature protein.

Neutrophil maturation in the bone marrow, granulopoiesis, is a tightly regulated process governed by a temporal and successive expression of transcription factors and appropriate stimulation by cytokines from the surrounding stromal tissue of the bone marrow. Observations published over the last decade have, however, made it increasingly clear that posttranscriptional fine-tuning of protein expression by microRNAs (miRNAs) also plays a very important role in this process. In this chapter the influence of miRNAs on normal neutrophil development and biology is reviewed. The majority of miRNAs alter their expression profile at the point in granulopoiesis where proliferation ceases and terminal differentiation commences indicating a major role of miRNAs in fine-tuning of cell cycle regulatory proteins and transcription factors governing this transition. Some miRNAs are induced by external stimuli of the mature neutrophil and act to fine-tune the innate immune action of the cell.

The pattern of expressed genes defines the structure and functional status of cells. Currently, most methods used in gene expression studies depend on large numbers of cells. Thus, their application may be hampered by the heterogeneity of cell populations, and by the low numbers of cells obtainable from in vivo sources. Such drawbacks may be overcome by methods suitable for the profiling of gene expression at the single cell level. We studied whether polymerase chain reaction (PCR) products synthesized from individual cells by global amplification of messenger RNA (mRNA) were suitable as probes for gene expression analysis. For this purpose, cells were subjected to reverse transcription and PCR using sequence independent primers (SIP RT-PCR). The resultant cDNA products were radiolabeled and hybridized to cDNA clones arrayed on a nylon membrane by vacuum slot blotting (a method referred to as slot blot analysis). The SIP RT-PCR procedure was reproducible and allowed the detection of twofold changes in input RNA copies per cell (range: 80-10.000 copies of an in vitro transcribed poly(A)-tailed RNA/cell). Analysis of total RNA and amplified cDNA, obtained from neutrophil granulocytes and the promyelocytic HL-60 cell line, demonstrated comparable gene expression profiles as measured by Northern blot and slot blot analysis. Slot blot analysis of HL-60 cells indicated that individual cells from an apparently homogenous population have varying expression of specific transcripts, which all contribute to the mRNA phenotype of their population. Interestingly, the genes that were detected in some but not all individual HL-60 cells were those found to peak within 2 days of retinoic acid-induced granulocytic differentiation. This study demonstrates the potential of cDNA, synthesized from individual cells by global amplification of mRNA, as probes for cDNA arrays.

The function of the mature polymorphonuclear neutrophil is dependent on its granules, each with its characteristic content of proteins. The granule proteins are formed at different stages during maturation of neutrophils from myeloblasts to segmented cells. The regulation of granule protein expression is controlled by a number of transcription factors, many of which are also essential for commitment of multipotent hematopoietic stem cells to lineage-committed myeloid progenitor cells and for differentiation of these progenitor cells; among these, PU.1 and C/EBPalpha stand out as critical for all granule proteins whereas AML-1 is critical for primary granule protein expression and C/EBPepsilon for secondary and tertiary granule protein expression.

The neutrophilic granulocyte is the most numerous leukocyte in peripheral blood. The development from a multipotent progenitor cell to a mature neutrophil takes place in the bone marrow over a period of 10-14 days. In order to understand the cellular mechanisms behind this process, it is necessary to investigate cells from different stages of neutrophil differentiation. As no human cell line has the ability to faithfully reproduce the entire differentiation process from promyelocyte to segmented neutrophil the analysis of many maturation-dependent processes has to be done on neutrophil precursors from human bone marrow. For this purpose, a technique whereby neutrophil precursors can be isolated from the bone marrow and separated according to their maturity is required. Two different methods have been shown to be useful for isolation of immature neutrophils: density centrifugation on a Percoll gradient, where the increasing density of the cells with maturity forms the basis of the separation, and multidimensional flow cytometry, where a combination of size, granulation, and surface markers are used for the discrimination of different neutrophil precursors. This paper will review these two methods for separation of neutrophil precursors with special emphasis on Percoll density centrifugation and the use of cells isolated by this technique for the analysis of neutrophil-specific mRNAs and the biosynthesis of neutrophil granule proteins.

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa lipocalin first identified as a protein stored in specific granules of the human neutrophil. The protein is believed to bind small lipophilic substances such as bacterial derived formylpeptides and lipopolysaccharides (LPS) and might function as a modulator of inflammation. To characterize the regulation of NGAL further, we have cloned and sequenced a 5869-bp region of the NGAL gene including 1695 bp of the 5' nontranscribed region and a 3696-bp coding region encompassing seven exons and six introns. The transcriptional start sites were identified by an RNase protection assay. The NGAL gene is highly homologous to the mouse gene 24p3. NGAL was expressed in bone marrow and in tissues that are prone to exposure to microorganisms. Potential cis-acting elements were identified in the promoter region of the NGAL gene by computer analysis and include binding sites for CTF/CBP, the hematopoietic transcription factors GATA-1 and PU.1, and the LPS-inducible factor NF-kappa B.

hCAP-18 is a newly described protein of human neutrophilic granulocytes which belongs to the cathelicidin family of antimicrobial proteins. Members of this protein family share a common N-terminal sequence followed by a highly diverse antimicrobial, cationic C-terminus. The present work describes the production of recombinant hCAP-18, the generation of antibodies to the protein and the development of an accurate, sensitive and specific ELISA for the detection of hCAP-18 in cells, plasma and urine with a detection limit of 0.084 ng/ml. The amount of hCAP-18 in neutrophils is 0.627 microgram protein per 10(6) cells. The plasma level is 1.18 micrograms/ml which is several fold higher than for other neutrophil specific granule proteins. hCAP-18 is present in plasma as high molecular weight complexes. In accordance with this, hCAP-18 is barely excreted in the urine. The bone marrow appears to be the major source of plasma hCAP-18. The high level of hCAP-18 in plasma may provide an important defense against microorganisms and endotoxins.

POLYMORPHONUCLEAR leukocytes were discovered by Paul Ehrlich,[1][1] when fixation and staining techniques made it possible to identify the lobulated nucleus and the granules that have given name to these cells and allowed for their classification as eosinophils, basophils, and neutrophils.

A novel system for heterologous expression of prohormones based on transient transfection of the HIT beta-cell line was established using human progastrin as a model. Progastrin was expressed at high levels compared to other gene transfer systems in endocrine cells, and the processing pattern was similar to that of normal antral gastrin cells. Thus, gastrin was partially tyrosine O-sulfated and carboxyamidated. Cell extracts contained mainly gastrin-17 and gastrin-34 and the corresponding glycine-extended forms. In contrast, the media contained more incompletely processed gastrin forms. This suggests that gastrin was directed to the regulated secretory pathway but that some progastrin products were constitutively secreted. Glucose increased both the level of gastrin gene expression and maturation to carboxyamidated peptides, indicating that glucose influences the activity of the amidation enzyme complex, peptidylglycine alpha-amidating mono-oxygenase (PAM), in insulin cells. Mutational analysis of tyrosine sulfation of gastrin demonstrated that substitution of the uncharged residue carboxy-terminal to the tyrosine with an acidic residue does not increase sulfation in contrast to previous results, where the amino-terminal residue was replaced with an acidic residue. The mutant peptides displayed sulfation-dependent processing, supporting our recent suggestion that tyrosine sulfation increases the proteolytic processing of prohormones.

Jumonji Domain-Containing Protein 3 (JMJD3)/lysine demethylase 6B (KDM6B) is an epigenetic modulator that removes repressive histone marks on genes. Expression of KDM6B mRNA is elevated in leukocytes from patients with ANCA-associated vasculitis (AAV) and has been suggested to be the reason for higher proteinase 3 (PR3) mRNA expression in these cells due to derepression of PRTN3 gene transcription. MicroRNA-941 (miR-941) has been shown to target KDM6B mRNA and inhibit JMJD3 production. We therefore investigated whether polymorphonuclear granulocytes (PMNs) from patients suffering from granulomatosis with polyangiitis (GPA) have lower expression of miR-941 than healthy control donors as a biological cause for higher JMJD3 levels. We found no significant difference in the degree of maturation of PMNs from GPA patients (n = 8) and healthy controls (n = 11) as determined from cell surface expression of the neutrophil maturation marker CD16 and gene expression profile of FCGR3B. The expression of PRTN3 and KDM6B mRNAs and miR-941 was not significantly different in GPA patients and healthy controls. Transfection of pre-miR-941 into the neutrophil promyelocyte cell line PLB-985 cells did not result in reduction of the KDM6B mRNA level as shown previously in a hepatocellular carcinoma cell line. The amount of PR3 in PMNs from GPA patients and healthy controls was comparable. In conclusion, we found that PRTN3 mRNA, KDM6B mRNA, and miR-941 expression levels in PMNs do not differ between GPA patients and healthy controls, and that miR-941 does not uniformly regulate KDM6B mRNA levels by inducing degradation of the transcript. Thus, decreased miR-941 expression in PMNs cannot be part of the pathogenesis of GPA.

NGAL/lipocalin-2 is a siderophore-binding protein that is highly expressed in several cancers. It is suggested to confer a proliferative advantage to cancer cells. Its expression has been correlated with aggressiveness of breast cancer as determined both in patients and in mouse breast cancer models. This was recently confirmed in two mouse models of spontaneous breast cancer in wild-type and lipocalin-2-deficient mice. We used a similar strategy using a different mouse strain. Lipocalin-2-deficient mice and mouse mammary tumor virus-polyoma middle T antigen (MMTV-PyMT) mice were crossed into the same FVB/N background. All mice developed tumors by week 8. The mice were sacrificed on week 13 and tissue was processed for biochemical and histological analysis. The total tumor volume and number of metastases were quantitated in 26 lipocalin-2-deficient mice and 34 wild-type controls. Lipocalin-2 expression in tumors of MMTV-PyMT-positive and wild-type mice was assessed by quantitative real-time PCR and by immunohistochemistry. The expression of the lipocalin-2 receptors 24p3R and megalin and of Mmp-9, transferrin receptor, and Bdh2 (a producer of a mammalian siderophore) were quantitated by real-time PCR. No significant difference was observed between wild-type and lipocalin-2-deficient mice. Lipocalin-2 was highly expressed in tumors from wild-type mice, but the expression did not correlate with tumor size. No effect of lipocalin-2 was observed with respect to time to tumor appearance, total tumor volume, or to the number of metastases. Histology and gelatinolytic activity of the mammary tumors did not differ between wild-type and lipocalin-2-deficient mice. We conclude that NGAL/lipocalin-2 does not invariably affect the aggressiveness of breast cancers as assessed in mouse models, thus questioning the role of lipocalin-2 in cancer development. publishedVersion

We investigated the DNA restriction fragment length polymorphism (RFLP) of the major histocompatibility complex (MHC) genes: HLA-DRB, -DQA, -DQB, -DPB in 24 Danish patients with systemic lupus erythematosus (SLE) and in 102 healthy Danes. A highly significant increase of the frequency of the DR3- and DRw6-associated 7.00 kb DRB TaqI DNA fragment was found in SLE patients compared to normal controls (83.3% vs 35.5%; RR = 9.1, p < 10(-4). The frequencies of the DQA1*0501-associated 4.56 kb DQA TaqI fragment and the DRB3*01/03-associated 9.79 kb TaqI fragment were also found to be significantly increased in SLE patients (70.8% vs 29.7%; RR = 5.8, p < 10(-2) for the DQA fragment and 70.8% vs 36.1%; RR = 4.3, p < 0.05 for the DRB3 fragment). Less extensive and insignificant increases of the frequencies of the DR3-associated DQB and DPB fragments were observed. The frequencies of the DR2-associated DRB, DQA, and DQB fragments were comparable to those found in normal controls.

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.

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.

Neutrophil granules store proteins that are critically important for the neutrophil to move from the vascular bed to tissues and to kill microorganisms. This is illustrated in nature when individual proteins are deleted due to inherited mutations of their cognate genes, and such deficiencies result in the conditions leucocyte adhesion deficiency and chronic granulomatous disease. The granules of the neutrophil have traditionally been divided into two or three major types but are instead a continuum where several subtypes can be identified with differences in protein content and propensity for mobilization. This is explained by the 'targeting by timing hypothesis' which states that granules are filled with granule proteins that are synthesized at the time the granule is formed. The heterogeneity of granules arises because the synthesis of granule proteins is individually controlled and major differences exist in the timings of biosynthesis during granulocytopoiesis. This is largely controlled by gene transcription.