Applying label-free dynamic mass redistribution assay for studying endogenous FPR1 receptor signalling in human neutrophils.

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 (pEC50) 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.

Ficolins do not alter host immune responses to lipopolysaccharide-induced inflammation in vivo.

Ficolins are a family of pattern recognition molecules that are capable of activating the lectin pathway of complement. A limited number of reports have demonstrated a protective role of ficolins in animal models of infection. In addition, an immune modulatory role of ficolins has been suggested. Yet, the contribution of ficolins to inflammatory disease processes remains elusive. To address this, we investigated ficolin deficient mice during a lipopolysaccharide (LPS)-induced model of systemic inflammation. Although murine serum ficolin was shown to bind LPS in vitro, there was no difference between wildtype and ficolin deficient mice in morbidity and mortality by LPS-induced inflammation. Moreover, there was no difference between wildtype and ficolin deficient mice in the inflammatory cytokine profiles after LPS challenge. These findings were substantiated by microarray analysis revealing an unaltered spleen transcriptome profile in ficolin deficient mice compared to wildtype mice. Collectively, results from this study demonstrate that ficolins are not involved in host response to LPS-induced systemic inflammation.

Changes in Gene Expression during G-CSF-Induced Emergency Granulopoiesis in Humans.

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 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 investigated in humans. In this work, we examine the changes in mRNA expression induced by administration of G-CSF in vivo, as a model of emergency granulopoiesis in humans. Blood samples were collected from healthy individuals after 5 d of G-CSF administration. Neutrophil precursors were sorted into discrete stages of maturation by flow cytometry, and RNA was subjected to microarray analysis. mRNA levels were compared with previously published expression levels in corresponding populations of neutrophil precursors isolated from bone marrow of untreated, healthy individuals. One thousand one hundred and ten mRNAs were differentially expressed >2-fold throughout terminal granulopoiesis. Major changes were seen in pathways involved in apoptosis, cytokine signaling, and TLR pathways. In addition, G-CSF treatment reduced the levels of four of five measured granule proteins in mature neutrophils, including the proantibacterial protein hCAP-18, which was completely deficient in neutrophils from G-CSF-treated donors. These results indicate that multiple biological processes are altered to satisfy the increased demand for neutrophils during G-CSF-induced emergency granulopoiesis in humans.

Granulopoiesis and granules of human neutrophils.

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.

miR-18b overexpression identifies mantle cell lymphoma patients with poor outcome and improves the MIPI-B prognosticator.

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.

Liver is the major source of elevated serum lipocalin-2 levels after bacterial infection or partial hepatectomy: a critical role for IL-6/STAT3.

UNLABELLED: 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.

Human α-defensin expression is not dependent on CCAAT/enhancer binding protein-ε in a murine model.

Specific granule deficiency (SGD) is a rare congenital disorder characterized by recurrent infections. The disease is caused by inactivating mutations of the CCAAT/enhancer binding protein-ε (C/EBP-ε) gene. As a consequence, specific and gelatinase granules lack most matrix proteins. Furthermore, azurophil granules contain diminished amounts of their most abundant proteins, α-defensins, also known as human neutrophil peptides (HNPs). In accordance with this, in vitro models have demonstrated induction of HNPs by C/EBP-ε. Since mice do not express myeloid defensins, they cannot per se be used to characterize the role of C/EBP-ε in controlling HNP expression in vivo. We therefore crossed a transgenic HNP-1-expressing mouse with the Cebpe-/- mouse to study the in vivo significance of C/EBP-ε for HNP-1 transcription and expression. Surprisingly, neither expression nor processing of HNP-1 was affected by lack of C/EBP-ε in these mice. Transduction of C/EBP-ε into primary bone marrow cells from HNP-1 mice induced some HNP-1 expression, but not to levels comparable to expression human cells. Taken together, our data infer that the HNP-1 of the transgenic mouse does not show an expression pattern equivalent to endogenous secondary granule proteins. This limits the use of these transgenic mice as a model for human conditions.

miRNA-130a regulates C/EBP-ε expression during granulopoiesis.

CCAAT/enhancer binding protein-ε (C/EBP-ε) is considered a master transcription factor regulating terminal neutrophil maturation. It is essential for expression of secondary granule proteins, but it also regulates proliferation, cell cycle, and maturation during granulopoiesis. Cebpe(-/-) mice have incomplete granulocytic differentiation and increased sensitivity toward bacterial infections. The amount of C/EBP-ε messenger RNA (mRNA) increases with maturation from myeloblasts with peak level in myelocytes (MC)/metamyelocytes (MM), when the cells stop proliferating followed by a decline in more mature cells. In contrast, C/EBP-ε protein is virtually detectable only in the MC/MM population, indicating that expression in more immature cells could be inhibited by microRNAs (miRNAs). We found that miRNA-130a (miR-130a) regulates C/EBP-ε protein expression in both murine and human granulocytic precursors. Overexpression of miR-130a in a murine cell line downregulated C/EBP-ε protein and lactoferrin (Ltf), cathelicidin antimicrobial protein (Camp), and lipocalin-2 (Lcn2) mRNA expression giving rise to cells with a more immature phenotype, as seen in the Cebpe(-/-) mouse. Introduction of a C/EBP-ε mRNA without target site for miR-130a restored both C/EBP-ε production, expression of Camp and Lcn2, and resulted in the cells having a more mature phenotype. We conclude that miR-130a is important for the regulation of the timed expression of C/EBP-ε during granulopoiesis.

MicroRNA profiling in human neutrophils during bone marrow granulopoiesis and in vivo exudation.

The purpose of this study was to describe the microRNA (miRNA) expression profiles of neutrophils and their precursors from the initiation of granulopoiesis in the bone marrow to extravasation and accumulation in skin windows. We analyzed three different cell populations from human bone marrow, polymorphonuclear neutrophil (PMNs) from peripheral blood, and extravasated PMNs from skin windows using the Affymetrix 2.0 platform. Our data reveal 135 miRNAs differentially regulated during bone marrow granulopoiesis. The majority is differentially regulated between the myeloblast/promyelocyte (MB/PM) and myelocyte/metamyelocyte (MC/MM) stages of development. These 135 miRNAs were divided into six clusters according to the pattern of their expression. Several miRNAs demonstrate a pronounced increase or reduction at the transition between MB/PM and MC/MM, which is associated with cell cycle arrest and the initiation of terminal differentiation. Seven miRNAs are differentially up-regulated between peripheral blood PMNs and extravasated PMNs and only one of these (miR-132) is also differentially regulated during granulopoiesis. The study indicates that several different miRNAs participate in the regulation of normal granulopoiesis and that miRNAs might also regulate activities of extravasated neutrophils. The data present the miRNA profiles during the development and activation of the neutrophil granulocyte in healthy humans and thus serves as a reference for further research of normal and malignant granulocytic development.

No effect of NGAL/lipocalin-2 on aggressiveness of cancer in the MMTV-PyMT/FVB/N mouse model for breast cancer.

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.

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.

Serglycin participates in retention of α-defensin in granules during myelopoiesis.

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.

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.

Lipocalin 2 is protective against E. coli pneumonia.

BACKGROUND: Lipocalin 2 is a bacteriostatic protein that binds the siderophore enterobactin, an iron-chelating molecule produced by Escherichia coli (E. coli) that is required for bacterial growth. Infection of the lungs by E. coli is rare despite a frequent exposure to this commensal bacterium. Lipocalin 2 is an effector molecule of the innate immune system and could therefore play a role in hindering growth of E. coli in the lungs. METHODS: Lipocalin 2 knock-out and wild type mice were infected with two strains of E. coli. The lungs were removed 48 hours post-infection and examined for lipocalin 2 and MMP9 (a myeloid marker protein) by immunohistochemical staining and western blotting. Bacterial numbers were assessed in the lungs of the mice at 2 and 5 days after infection and mortality of the mice was monitored over a five-day period. The effect of administering ferrichrome (an iron source that cannot be bound by lipocalin 2) along with E.coli was also examined. RESULTS: Intratracheal installation of E. coli in mice resulted in strong induction of lipocalin 2 expression in bronchial epithelium and alveolar type II pneumocytes. Migration of myeloid cells to the site of infection also contributed to an increased lipocalin 2 level in the lungs. Significant higher bacterial numbers were observed in the lungs of lipocalin 2 knock-out mice on days 2 and 5 after infection with E. coli (p < 0.05). In addition, a higher number of E. coli was found in the spleen of surviving lipocalin 2 knock-out mice on day 5 post-infection than in the corresponding wild-type mice (p < 0.05). The protective effect against E. coli infection in wild type mice could be counteracted by the siderophore ferrichrome, indicating that the protective effect of lipocalin 2 depends on its ability to sequester iron. CONCLUSIONS: Lipocalin 2 is important for protection of airways against infection by E. coli.

Induction of neutrophil gelatinase-associated lipocalin expression by co-stimulation with interleukin-17 and tumor necrosis factor-alpha is controlled by IkappaB-zeta but neither by C/EBP-beta nor C/EBP-delta.

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.

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.

Serglycin proteoglycan is not implicated in localizing exocrine pancreas enzymes to zymogen granules.

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) 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.