Stine N. Clemmensen

Papillon-Lefèvre syndrome patient reveals species-dependent requirements for neutrophil defenses.

Papillon-Lefèvre syndrome (PLS) results from mutations that inactivate cysteine protease cathepsin C (CTSC), which processes a variety of serine proteases considered essential for antimicrobial defense. Despite serine protease-deficient immune cell populations, PLS patients do not exhibit marked immunodeficiency. Here, we characterized a 24-year-old woman who had suffered from severe juvenile periodontal disease, but was otherwise healthy, and identified a homozygous missense mutation in CTSC indicative of PLS. Proteome analysis of patient neutrophil granules revealed that several proteins that normally localize to azurophil granules, including the major serine proteases, elastase, cathepsin G, and proteinase 3, were absent. Accordingly, neutrophils from this patient were incapable of producing neutrophil extracellular traps (NETs) in response to ROS and were unable to process endogenous cathelicidin hCAP-18 into the antibacterial peptide LL-37 in response to ionomycin. In immature myeloid cells from patient bone marrow, biosynthesis of CTSC and neutrophil serine proteases appeared normal along with initial processing and sorting to cellular storage. In contrast, these proteins were completely absent in mature neutrophils, indicating that CTSC mutation promotes protease degradation in more mature hematopoietic subsets, but does not affect protease production in progenitor cells. Together, these data indicate CTSC protects serine proteases from degradation in mature immune cells and suggest that neutrophil serine proteases are dispensable for human immunoprotection.

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.

Subcellular Fractionation of Human Neutrophils and Analysis of Subcellular Markers

The neutrophil has long been recognized for its impressive number of cytoplasmic granules that harbor proteins indispensable for innate immunity. Analysis of isolated granules has provided important information on how the neutrophil grades its response to match the challenges it meets on its passage from blood to tissues. Nitrogen cavitation was developed as a method for disruption of cells on the assumption that sudden reduction of the partial pressure of nitrogen would lead to aeration of nitrogen dissolved in the lipid bilayer of plasma membranes. We find that cells are broken by the shear stress that is associated with passage through the outlet valve under high pressure and that this results in disruption of the neutrophil cell membrane while granules remain intact. The unique properties of Percoll as a sedimentable density medium with no inherent tonicity or viscosity are used for creation of continuous density gradients with shoulders in the density profile created to optimize the physical separation of granule subsets and light membranes. Immunological methods (sandwich enzyme-linked immunosorbent assays) are used for quantitation of proteins that are characteristic constituents of the granule subsets of neutrophils.

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.

Plasma levels of OLFM4 in normals and patients with gastrointestinal cancer

Olfactomedin 4 (OLFM4) is a secreted glycoprotein predominantly expressed in bone marrow and gastrointestinal tissues. Aberrant expression of OLFM4 has been shown in several cancers. However, the clinical significance hereof is currently controversial. OLFM4 has been proposed as a candidate biomarker of gastrointestinal cancers. To address this, we developed monoclonal antibodies against synthetic peptides representing various segments of OLFM4. We examined expression of OLFM4 in epithelial cells by immunohistochemistry and found that OLFM4 is highly expressed in proliferating benign epithelial cells and in some carcinoma cells. We developed an Enzyme Linked Immunosorbent Assay for OLFM4 and investigated whether plasma levels of OLFM4 reflect colorectal malignancies, but were unable to see any such association. Instead, we observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 μg/ml, mean 0.028 μg/ml while 10% of both normals and patients with cancers had OLFM4 between 4 and 60 μg/ml, mean 15 μg/ml. The levels were constant over time. The background for this high plasma level is not known, but must be taken into account if OLFM4 is used as biomarker for GI cancers.

Non-invasive imaging of retinal blood flow in myeloproliferative neoplasms.

To study the circulation in the retinal vessels in patients with blood dyscrasia due to myeloproliferative neoplasms using non‐invasive retinal imaging.