Maria Torp Larsen

Olfactomedin 4 defines a subset of human neutrophils.

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

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

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

NSP4 Is Stored in Azurophil Granules and Released by Activated Neutrophils as Active Endoprotease with Restricted Specificity

Whereas neutrophil elastase, cathepsin G, and proteinase 3 have been known as granule-associated serine proteases of neutrophils for decades, a fourth member, called neutrophil serine protease 4 (NSP4), was just recently described and provisionally characterized. In this study, we identified NSP4 as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions as well as by RT-PCR analyses of neutrophil precursors from human bone marrow. The highest mRNA levels were observed in myeloblasts and promyelocytes, similar to myeloperoxidase, a marker of azurophil granules. To determine the extended sequence specificity of recombinant NSP4, we used an iterative fluorescence resonance energy transfer–based optimization strategy. In total, 142 different peptide substrates with arginine in P1 and variations at the P1′, P2′, P3, P4, and P2 positions were tested. This enabled us to construct an α1-proteinase inhibitor variant (Ile-Lys-Pro-Arg−/−Ser-Ile-Pro) with high specificity for NSP4. This tailor-made serpin was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils, indicating that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. Moreover, cathepsin C was identified as the activator of NSP4 in vivo, as cathepsin C deficiency resulted in a complete absence of NSP4 in a Papillon-Lefèvre patient. Our in-depth analysis of NSP4 establishes this arginine-specific protease as a genuine member of preactivated serine proteases stored in azurophil granules of human neutrophils.

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.

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.

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.

Differential role of AMPA receptors in mouse tests of antidepressant and anxiolytic action.

Depression and anxiety often co-occur, and conventional monoamine-facilitating antidepressants show efficacy against symptoms in both disorders. Rodent studies indicate that antidepressant effects of monoamine-based antidepressants involve increased α-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid glutamate receptor (AMPAR) neurotransmission, and positive allosteric modulators (PAMs) at AMPARs produced antidepressant-like effects in rodents. While this suggests that increased AMPAR-mediated neurotransmission is beneficial in depression management, preclinical studies addressing AMPARs in relation to anxiety have given ambiguous results with both anxiolytic-like and anxiogenic-like effects observed after AMPAR blockade. This study systematically compared the effects of the AMPAR potentiator LY451646 and the AMPAR antagonist GYKI-53655 on depression-related behaviour using the mouse forced swim (FST) and tail suspension tests (TST), and anxiety-related behaviour using the elevated zero maze (EZM), marble burying (MB) and novelty-induced hypophagia (NIH) tests. The serotonin-selective antidepressant citalopram was included for comparison. Due to the importance of AMPARs in learning and memory we also tested if GYKI-53655 disrupted performance in the V-maze test for attention-dependent behaviour, and the social transmission of food preference (STFP) test of long-term memory. LY451646 (3 mg/kg) showed an antidepressant-like profile in the FST and TST, and GYKI-53655 (≥ 5 mg/kg) had a depressogenic-like effect in the TST but no effect in the FST. Conversely, GYKI-53655 produced marked anxiolytic-like effects in the EZM (≥ 2.5 mg/kg), MBT (≥ 2.5 mg/kg), and NIH tests (≥ 5 mg/kg), while LY451646 (≥ 3 mg/kg) increased anxiety-like behaviour in the EZM. Citalopram showed an antidepressant-like effect in the FST (≥ 10 mg/kg), but not TST, an anxiolytic-like effect in the EZM (≥ 3 mg/kg) and MB test (≥ 2.5 mg/kg), and an anxiogenic-like effect in the NIH test (≥ 30 mg/kg). GYKI-53655 did not affect cognitive performance in the V-maze or STFP tests. Collectively, these findings suggest a differential role of AMPARs in depression and anxiety, with AMPAR activation promoting antidepressant responses and AMPAR inhibition promoting anxiolytic responses. The potential of AMPARs as a novel target in depression and anxiety pharmacotherapy is discussed.

Positive allosteric modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors differentially modulates the behavioural effects of citalopram in mouse models of antidepressant and anxiolytic action.

Drugs that increase monoamine neurotransmission are effective in both anxiety and depression. The therapeutic effects of monoamine-based antidepressant drugs may involve indirect effects on neurotransmission through &agr;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR). Thus, chronic antidepressant treatment increases AMPAR-mediated neurotransmission and AMPAR-positive allosteric modulators have shown antidepressant-like efficacy in rodents. Here, the effect of enhanced AMPAR neurotransmission on the antidepressant-like and anxiolytic-like actions of the selective serotonin reuptake inhibitor citalopram (0–10 mg/kg) was investigated in mice using the AMPAR-positive allosteric modulator LY451646 (0–3 mg/kg). Antidepressant-like effects were assessed using the forced-swim test (FST), whereas anxiolytic-like effects were tested using the elevated zero maze (EZM) and the marble burying test. LY451646 (3 mg/kg) increased swim distance in the FST and a subactive dose of LY451646 (1 mg/kg) enhanced the effect of citalopram in the FST. In the EZM, LY451646 (3 mg/kg) did not show anxiogenic effects alone, but blocked the anxiolytic-like action of citalopram in the EZM, as reflected by an increase in the latency to enter the open areas and a decrease in the number of entries and time spent in the open areas in citalopram-treated mice. In the marble burying test, LY451646 (3 mg/kg) showed no effect alone, but significantly attenuated the anxiolytic-like effect of citalopram (1.25–2.5 mg/kg) by increasing the number of marbles buried in citalopram-treated mice. These results suggest that AMPAR neurotransmission plays opposite roles in anxiety and depression as AMPAR potentiation facilitated the antidepressant-like effects of citalopram while attenuating its anxiolytic-like effect. These findings have ramifications in the search for AMPAR-based novel anxiolytic and antidepressant treatments.

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.