Variation in FCN1 affects biosynthesis of ficolin-1 and is associated with outcome of systemic inflammation.

Ficolin-1 is a recognition molecule of the lectin complement pathway. The ficolin-1 gene FCN1 is polymorphic, but the functional and clinical consequences are unknown.The concentration of ficolin-1 in plasma and FCN1 polymorphisms in positions -1981 (rs2989727), -791 (rs28909068), -542 (rs10120023), -271 (rs28909976), -144 (rs10117466) and +7918 (rs1071583) were determined in 100 healthy individuals. FCN1 expression by isolated monocytes and granulocytes and ficolin-1 levels in monocyte culture supernatants were assessed in 21 FCN1-genotyped individuals. FCN1 polymorphisms were determined in a cohort of 251 patients with systemic inflammation. High ficolin-1 plasma levels were significantly associated with the minor alleles in position -542 and -144. These alleles were also significantly associated with high FCN1 mRNA expression. The level of ficolin-1 in culture supernatants was significantly higher in individuals homozygous for the minor alleles at positions -542 and -144. Homozygosity for these alleles was significantly associated with fatal outcome in patients with systemic inflammation. None of the other investigated polymorphisms were associated with FCN1 and ficolin-1 expression, concentration or disease outcome. Functional polymorphic sites in the promoter region of FCN1 regulate both the expression and synthesis of ficolin-1 and are associated with outcome in severe inflammation.

Neutrophil granules in health and disease.

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.

The exocytotic fusion pore of small granules has a conductance similar to an ion channel.

We measured capacitance changes in cell attached patches of human neutrophils using a high frequency lock-in method. With this technique the noise level is reduced to 0.025 fF such that capacitance steps of 0.1 fF are clearly detected corresponding to exo- and endocytosis of single 60 nm vesicles. It is thus possible to detect almost all known exocytotic and endocytotic processes including exocytosis of small neurotransmitter containing vesicles in most cell types as well as endocytosis of coated and uncoated pits. In neutrophils we demonstrate a stepwise capacitance decrease generated by 60-165 nm vesicles as expected for endocytosis of coated and non-coated pits. Following ionomycin stimulation a stepwise capacitance increase is observed consisting of 0.1-5 fF steps corresponding to the different granule types of human neutrophils from secretory vesicles to azurophil granules. The opening of individual fusion pores is resolved during exocytosis of 200 nm vesicles. The initial conductance has a mean value of 150 pS and can be as low as 35 pS which is similar to the conductance of many ion channels suggesting that the initial fusion pore is formed by a protein complex.

Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation.

We describe a new method for subcellular fractionation of human neutrophils. Neutrophils were disrupted by nitrogen cavitation and the nuclei removed by centrifugation. The postnuclear supernatant was applied on top of a discontinuous Percoll density gradient. Centrifugation for 15 min at 48,000 g resulted in complete separation of plasma membranes, azurophil granules, and specific granules. As determined by ultrastructure and the distribution of biochemical markers of these organelles, approximately 90% of the b-cytochrome in unstimulated cells was recovered from the band containing the specific granules and was shown to be in or tightly associated with the membrane. During stimulation of intact neutrophils with phorbol myristate acetate or the ionophore A23187, we observed translocation of 40-75% of the b-cytochrome to the plasma membrane. The extent of this translocation closely paralleled release of the specific granule marker, vitamin B12-binding protein. These data indicate that the b-cytochrome is in the membrane of the specific granules of unstimulated neutrophils and that stimulus-induced fusion of these granules with the plasma membrane results in a translocation of the cytochrome. Our observations provide a basis for the assembly of the microbicidal oxidase of the human neutrophil.

Chemoattractant-regulated mobilization of a novel intracellular compartment in human neutrophils.

A novel mobilizable intracellular compartment was identified in human neutrophils by latent alkaline phosphatase activity. This compartment is mobilized to the plasma membrane much more readily than any identified granule subset and has kinetics of up-regulation in the membrane similar to those reported for a variety of receptor proteins. Triton X-100 permeabilization of both intact human neutrophils and subcellular fractions obtained by density-gradient centrifugation revealed that 70 percent of the alkaline phosphatase is located in an intracellular compartment distinct from primary, secondary, and gelatinase granules and from the plasma membrane. This compartment fully translocates to the plasma membrane after stimulation with nanomolar concentrations of the chemotactic peptide N-formylmethionylleucylphenylalanine.

Role of oxygen in antibody-dependent cytotoxicity mediated by monocytes and neutrophils.

The antibody-dependent cell-mediated cytoxicity (ADCC) by human monocytes and neutrophils was investigated by measuring the release of 51chromate from prelabeled erythrocytes coated with immunoglobulin G. ADCC was found to be positively correlated to phagocytosis of 51Cr-labeled erythrocytes and to the postphagocytic events of the effector cells, activation of the hexose monophosphate shunt, and degranulation. Exclusion of oxygen from the incubation media halved the ADCC by both cell types without affectijg phagocytosis or degranulation. Likewise, ADCC by cells from patients suffering from chronic granulomatous disease (CGD) was only half the intensity of ADCC by cells from normals. Inhibitors of mitochondrial respiration were without depressing effect of ADCC. Azide, which in addition to its blocking action on oxydative phosphorylation also inhibits catalase and myeloperoxidase, resulted in a approximately equal to 40% stimulation of ADCC by cells from normals but was without effect of ADCC by cells from CGD patients. The hydroxyl radical scavenger, mannitol, significantly depressed ADCC by cells from normals (P < 0.01) but was without effect on cells from CGD patients. Azide and mannitol also were without effect on ADCC by normal cells when oxygen was excluded. In a xanthine-xanthine oxidase system, erythrocytes were effectively lysed. This lysis was inhibited by catalase, superoxide dismutase, and mannitol. When comparable concentrations of glucose oxidase were used no lysis was observed. H2O2 either alone or in combination with azide did not lyse erythrocytes. It is suggested that ADCC by both monocytes and neutrophils is partly dependent on the generation of hydroxyl radicals by the effector cells.

Energy metabolism of human neutrophils during phagocytosis.

Detailed quantitative studies were performed on the generation and utilization of energy by resting and phagocytosing human neutrophils. The ATP content was 1.9 fmol/cell, was constant during rest, and was not influenced by the presence or absence of glucose in the medium. The intracellular content of phosphocreatine was less than 0.2 fmol/cell. In the presence of glucose, ATP was generated almost exclusively from lactate produced from glucose taken up from the surrounding medium. The amount of lactate produced could account for 85% of the glucose taken up by the cells, and the intracellular glycosyl store, glycogen, was not drawn upon. The rate of ATP generation as calculated from the rate of lactate production was 1.3 fmol/cell/min. During phagocytosis, there was no measurable increase in glucose consumption or lactate production, and the ATP content fell rapidly to 0.8 fmol/cell. This disappearance of ATP was apparently irreversible since no corresponding increase in ADP or AMP was observed. It therefore appears that this phagocytosis-induced fall in ATP concentration represents all the extra energy utilized in human neutrophils in the presence of glucose. In the absence of glucose, the rate of ATP generation in the resting cell was considerably smaller, 0.75 fmol/cell per min, as calculated from the rate of glycolysis, which is sustained exclusively by glycogenolysis. Under this condition, however, phagocytosis induces significant enhancement of glycogenolysis and the rate of lactate production is increased by 60%, raising the rate of ATP generation to 1.2 fmol/cell per min. Nonetheless, the ATP content drops significantly from 1.9 to 1.0 fmol/cell. Neutrophils from patients with chronic granulomatous disease have the same rate of glycolysis and the same ATP content as normal cells, thus confirming that the defective respiration of these cells does not affect their energy metabolism.

Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst.

Phagocytosis by neutrophils is accompanied by a burst in O2 consumption and activation of the hexose monophosphate shunt (HMPS). Proton secretion equal to the amount of O2 consumed is an additional feature of the respiratory burst, but its source has not been identified, nor has the source of all electrons donated to O2 in the respiratory burst. We chemically quantitated total CO2 generation in human neutrophils and found that proton secretion elicited by phagocytosis was accompanied by a stoichiometric increase in CO2 generation. Addition of carbonic anhydrase and its inhibitors had no effect on either the quantities of CO2 measured or the quantities of protons secreted. Therefore, the CO2 generated in the respiratory burst of stimulated neutrophils is hydrated to form H2CO3, which then dissociates, accounting for the observed proton secretion. Furthermore, the CO2 generated corresponds to the O2 consumed with a respiratory quotient of nearly 1. We conclude on the basis of this and previous studies that the HMPS activity is the source of both the electrons for the NADPH oxidase and of protons secreted in association with the respiratory burst.

Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils.

The subcellular localization of Mac-1 was determined in resting and stimulated human neutrophils after disruption by nitrogen cavitation and fractionation on two-layer Percoll density gradients. Light membranes were further separated by high voltage free flow electrophoresis. Mac-1 was determined by an ELISA with monoclonal antibodies that were specific for the alpha-chain (CD11b). In unstimulated neutrophils, 75% of Mac-1 colocalized with specific granules including gelatinase granules, 20% with secretory vesicles and the rest with plasma membranes. Stimulation with nanomolar concentrations of FMLP resulted in the translocation of Mac-1 from secretory vesicles to the plasma membrane, and only minimal translocation from specific granules and gelatinase granules. Stimulation with PMA or Ionomycin resulted in full translocation of Mac-1 from secretory vesicles and gelatinase granules to the plasma membrane, and partial translocation of Mac-1 from specific granules. These findings were corroborated by flow cytometry, which demonstrated a 6-10-fold increase in the surface membrane content of Mac-1 in response to stimulation with FMLP, granulocyte-macrophage colony stimulating factor, IL-8, leukotriene B4, platelet-activating factor, TNF-alpha, and zymosan-activated serum, and a 25-fold increase in response to Ionomycin. Thus, secretory vesicles constitute the most important reservoir of Mac-1 that is incorporated into the plasma membrane during stimulation with inflammatory mediators.

Identification of a highly mobilizable subset of human neutrophil intracellular vesicles that contains tetranectin and latent alkaline phosphatase.

Tetranectin, a protein recently identified in a wide variety of human secretory cells (Christensen, L., and I. Clemmensen. 1989. Histochemistry. 92:29-35) was found to colocalize with latent alkaline phosphatase activity in fractions well separated from azurophil granules, specific granules, gelatinase-containing granules, and plasma membranes when postnuclear supernatants of nitrogen-cavitated neutrophils were fractionated on discontinuous Percoll density gradients. Stimulation of intact neutrophils with nanomolar concentrations of FMLP, leukotriene B4, 10-100 U/ml of tumor necrosis factor, and granulocyte-macrophage colony-stimulating factor resulted in parallel release of tetranectin and translocation of alkaline phosphatase to the plasma membrane. Furthermore, intracellular pools of tetranectin and latent alkaline phosphatase were completely released from neutrophils under conditions that barely induced release of specific granules containing B12-binding protein. These findings indicate that tetranectin and latent alkaline phosphatase define an easily mobilizable population of cytoplasmic storage organelles in human neutrophils which are functionally distinguishable from azurophil, specific, and gelatinase-containing granules. These organelles may play an important role as stores of membrane proteins that are mobilized to the cell surface during stimulation by inflammatory mediators.

Stimulated mobilization of monocyte Mac-1 and p150,95 adhesion proteins from an intracellular vesicular compartment to the cell surface.

Monocytes were stimulated to increase their cell surface quantity of leukocyte adhesion proteins p150,95 and Mac-1 by the chemoattractant formyl-methionyl-leucyl-phenylalanine, or other mediators such as platelet-derived growth factor, tumor necrosis factor, C5a, and leukotriene B4. Dose-response curves indicated variations in the sensitivity of monocytes and granulocytes to these mediators. These increases were independent of protein synthesis and half-maximal at 2 min. Human alveolar and murine peritoneal macrophages, cells that had previously diapedised, could not be induced to upregulate Mac-1 or p150,95. Detergent permeabilization studies in monocytes indicated that these proteins were stored in internal latent pools, which were reduced upon stimulation. Electron microscopy utilizing rabbit antiserum against p150,95 revealed these proteins on the plasma membrane, and in intracellular vesicles and peroxidase negative granules. Together with other functional studies, these findings suggest that the mobilization of Mac-1 and p150,95 from an intracellular compartment to the plasma membrane regulates the monocyte's ability to adhere and diapedese.

Stimulus-dependent secretion of plasma proteins from human neutrophils.

In search for matrix proteins released from secretory vesicles of human neutrophils, a prominent 67-kD protein was identified in the extracellular medium of neutrophils stimulated by the chemotactic peptide, FMLP. The protein was purified to apparent homogeneity and partially sequenced. The sequence of the first 32 NH2-terminal amino acids was identical to the sequence of albumin. mRNA for human albumin could not be detected in bone marrow cells, nor could biosynthetic labeling of albumin be demonstrated in bone marrow cells during incubation with [14C]leucine. Immunofluorescence studies on single cells demonstrated the presence of intracellular albumin in fixed permeabilized neutrophils. Light microscopy of immunogold-silver-stained cryosections visualized albumin in cytoplasmic "granules." The morphology of these was determined by immunoelectron microscopy as vesicles of varying form and size. Subcellular fractionation studies on unstimulated neutrophils demonstrated the presence of albumin in the low density pre-gamma and gamma-regions that contain secretory vesicles, but are devoid of specific granules and azurophil granules. Albumin was readily released from these structures during activation of neutrophils with inflammatory mediators. Immunoblotting demonstrated the presence of immunoglobulin and transferrin along with albumin in exocytosed material from stimulated neutrophils. This indicates that secretory vesicles are unique endocytic vesicles that can be triggered to exocytose by inflammatory stimuli.

Effect of glycogenolytic agents on glycogen synthase activity in polymorphonuclear leukocytes. Evidence for a Ca2+-mediated regulation of glycogen synthase activity.

Human polymorphonuclear leukocytes were found to respond to the beta-receptor activators, adrenalin and isoproterenol, with a rapid transient increase in cyclic AMP, activation of cyclic AMP-dependent protein kinase, phosphorylase kinase, deactivation of glycogen synthase and glycogen breakdown. This response was unaffected by the presence of 10 mM EGTA. Incubation of leukocytes with phorbol myristate acetate, which stimulates the hexose monophosphate shunt by a Ca2+ mediated mechanism, resulted in activation of phosphorylase without affecting cyclic AMP-dependent protein kinase or phosphorylase kinase activity, thus indicating a Ca2+-mediated activation of phosphorylase. This was, however, unaffected by EGTA. Prolonged incubation with phorbol myristate acetate was found to result in a parallel activation of phosphorylase and glycogen synthase secondary to a pronounced depletion of cellular glycogen. Addition of glucose to polymorphonuclear leukocytes resulted in total conversion of phosphorylase a to the b form and activation of glycogen synthase, however, when EGTA was included, the response to glucose was greatly amplified, thus indicating the synthase conversion is regulated by Ca2+ sensitive mechanisms which do not involve phosphorylase kinase. Addition of adrenalin to cells previously activated by glucose resulted in an increase in the concentration of cyclic AMP and activation of cyclic AMP-dependent protein kinase but deactivation of synthase was not effectuated under these conditions.

Interactions between neutrophil gelatinase-associated lipocalin and natural lipophilic ligands.

Neutrophils are activated by both paracrine molecules, e.g. platelet activating factor (PAF) and leukotriene B4 (LTB4), and the bacterial hydrophobic peptide N-formyl-Met-Leu-Phe (fMLP). Several mechanisms are involved in regulation of the activation, including receptor endocytosis and ligand breakdown. The interactions between the specific granule protein neutrophil gelatinase-associated lipocalin (NGAL), expressed in human neutrophils, and fMLP, PAF and LTB4, were investigated by weak affinity chromatography. NGAL was immobilised to a silica matrix and packed in a micro-column and the retention times of retarded ligands were measured and used to calculate the strength of the interactions. The association constants for fMLP were K(ass) = 0.85 x 10(3) M(-1) at 20 degrees C and 0.77 x 10(3) M(-1) at 37 degrees C, for LTB4 were K(ass) = 4.37 x 10(3) M(-1) at 20 degrees C and 3.27 x 10(3) M(-1) at 37 degrees C and for PAF were K(ass) = 25.4 x 10(3) M(-1) at 20 degrees C and 10.5 x 10(3) M(-1) at 37 degrees C. Other methods of detecting the interactions such as gel filtration, immunoprecipitation, photoactivated ligands and fluorescence quenching proved to be insufficient. The results demonstrate the superiority of weak affinity chromatography as a method of studying the interactions of the specific granule protein NGAL.

Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse.

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa lipocalin originally purified from human neutrophils. It exists in monomeric and homo- and heterodimeric forms, the latter as a dimer with human neutrophil gelatinase. It is secreted from specific granules of activated human neutrophils. Homologous proteins have been identified in mouse (24p3/uterocalin) and rat (alpha(2)-microglobulin-related protein/neu-related lipocalin). Structural data have confirmed a typical lipocalin fold of NGAL with an eight-stranded beta-barrel, but with an unusually large cavity lined with more polar and positively charged amino acid residues than normally seen in lipocalins. Chemotactic formyl-peptides from bacteria have been proposed as ligands of NGAL, but binding experiments and the structure of NGAL do not support this hypothesis. Besides neutrophils, NGAL is expressed in most tissues normally exposed to microorganisms, and its synthesis is induced in epithelial cells during inflammation. This may indicate either a microbicidal activity of NGAL or a role in regulation of inflammation or cellular growth, putative functions yet to be demonstrated.

Activation of the oxygen-radical-generating system in granules of intact human neutrophils by a calcium ionophore (ionomycin).

Subcellular fractionation studies were performed on human neutrophils stimulated with ionomycin (a Ca(2+)-specific ionophore). The results of these studies revealed NADPH-oxidase activity, without any additive, both in the plasma membrane and in the specific granule fractions. After comparing these results with the NADPH oxidase activity induced by the ionophore in intact neutrophils, in differentiated HL-60 cells and in neutrophil cytoplasts, we conclude that ionomycin preferentially activates the NADPH oxidase pool located in the membrane of specific granules. Furthermore, we suggest that incorporation of granule membrane into the plasma membrane makes the associated NADPH oxidase less sensitive to activation induced by a rise in [Ca(2+)]i.

Crystal structure of human grancalcin, a member of the penta-EF-hand protein family.

Grancalcin is a Ca(2+)-binding protein expressed at high level in neutrophils. It belongs to the PEF family, proteins containing five EF-hand motifs and which are known to associate with membranes in Ca(2+)-dependent manner. Prototypic members of this family are Ca(2+)-binding domains of calpain. Our recent finding that grancalcin interacts with L-plastin, a protein known to have actin bundling activity, suggests that grancalcin may play a role in regulation of adherence and migration of neutrophils. The structure of human grancalcin has been determined at 1.9 A resolution in the absence of calcium (R-factor of 0.212 and R-free of 0.249) and at 2. 5 A resolution in the presence of calcium (R-factor of 0.226 and R-free of 0.281). The molecule is predominantly alpha-helical: it contains eight alpha-helices and only two short stretches of two-stranded beta-sheets between the loops of paired EF-hands. Grancalcin forms dimers through the association of the unpaired EF5 hands in a manner similar to that observed in calpain, confirming this mode of association as a paradigm for the PEF family. Only one Ca(2+) was found per dimer under crystallization conditions that included CaCl(2). This cation binds to EF3 in one molecule, while this site in the second molecule of the dimer is unoccupied. This unoccupied site shows higher mobility. The structure determined in the presence of calcium, although does not represent a fully Ca(2+)-loaded form, suggests that calcium induces rather small conformational rearrangements. Comparison with calpain suggests further that the relatively small magnitude of conformational changes invoked by calcium alone may be a characteristic feature of the PEF family. Moreover, the largest differences are localized to the EF1, thus supporting the notion that calcium signaling occurs through this portion of the molecule and that it may involve the N-terminal Gly/Pro rich segment. Electrostatic potential distribution shows significant differences between grancalcin and calpain domain VI demonstrating their distinct character.

Patient education--a strategy for prevention of infections caused by permanent central venous catheters in patients with haematological malignancies: a randomized clinical trial.

A functioning tunnelled central venous catheter (CVC) is a crucial device for patients with haematological malignancies receiving high-dose intravenous chemotherapy. Despite the advantages, CVC infections are a major cause of sepsis and prolonged hospital stay. This study investigated the impact of patient education regarding provision of their own catheter care on the frequency of CVC-related infections (CRIs) and was conducted at a specialized haematological unit at the University Hospital of Copenhagen Rigshospitalet. From May to September 2002, 82 patients fitted with tunnelled double-lumen Hickman catheters were randomized consecutively. The intervention group (42 participants) received individualized training and supervision by a clinical nurse specialist, with the aim of becoming independently responsible for their own catheter care. The control group (40 participants) followed the standard CVC procedures carried out by nurses inside and outside the central hospital. A significant reduction in CRIs was found in the intervention group, with a >50% reduction in the incidence rate of CRIs. We conclude that systematic individualized, supervised patient education is able to reduce catheter-related infections.

A family with chronic haemolysis and selective accumulation of erythrocyte CDP-choline.

In this paper, we report a family with compensated chronic haemolysis where the only erythrocyte abnormality detected was an increased level of erythrocyte CDP-choline. Using 31P-NMR spectroscopy and enzymatic analysis the possibility of a pyrimidine 5'-nucleotidase deficiency was excluded. Thus, this family represents the first evidence for a hereditary haemolytic anaemia where the inferred enzymatic defect is located to choline phosphotransferase, the enzyme catalysing the final step in lecithin synthesis. The family history indicates an autosomal dominant mode of transmission with incomplete penetrance.