James E. Smolen

Human neutrophil annexin I promotes granule aggregation and modulates Ca(2+)-dependent membrane fusion.

The mechanism and cofactor requirements of exocytotic membrane fusion in neutrophils are unknown. Cytosolic proteins have been implicated in membrane fusion events. We assessed neutrophil cytosol for the presence of fusogenic proteins using a liposome fusion assay (lipid mixing). A fusogenic 36-kD protein containing amino acid sequence homology with human annexin I was purified from the cytosol of human neutrophils. This protein also shared functional characteristics with annexin I: it associated with and promoted lipid mixing of liposomes in a Ca(2+)-dependent manner at micromolar Ca2+ concentrations. The 36-kD protein required diacylglycerol to promote true fusion (contents mixing) at the same Ca2+ concentrations used for lipid mixing. The 36-kD protein exhibited a biphasic dose-response curve, by both promoting and inhibiting Ca(2+)-dependent lipid-mixing between liposomes and a plasma membrane fraction. The 36-kD protein also promoted Ca(2+)-dependent increases in aggregation of a specific granule fraction, as measured by a turbidity increase. Antiannexin I antibodies depleted the 36-kD protein from the cytosol by greater than 70% and diminished its ability to promote lipid mixing. Antiannexin I antibodies also decreased by greater than 75% the ability of neutrophil cytosol to promote Ca(2+)-dependent aggregation of the specific granules. These data suggest that annexin I may be involved in aggregation and fusion events in neutrophils.

Inositol trisphosphate mobilizes intracellular calcium in permeabilized adrenal chromaffin cells

Abstract: Using permeabilized chromaffin cells and the fluorescent probe Quin 2 (an indicator of free Ca2+), we found that inositol trisphosphate (IP3) specifically triggered an immediate and dose‐dependent release of Ca2+ from intracellular stores. Desensitization of the response was observed at nonsaturating concentrations of inositol trisphosphate and resequestration of Ca2+ was not observed. While representing only a small fraction of the total cellular Ca2+, the amount released by IP3 could significantly raise cytosolic Ca2+ and may account for muscarinic effects on Ca2+ metabolism in chromaffin cells.

Identification of glyceraldehyde-3-phosphate dehydrogenase as a Ca2+-dependent fusogen in human neutrophil cytosol.

The membrane fusion events observed during neutrophil degranulation are important aspects of the immunoregulatory system. In an attempt to understand the regulation of granule‐plasma membrane fusion, we have begun characterizing human neutrophil cytosol for fusion activity, finding that 50% of the fusogenic activity could be attributed to members of the annexin family of proteins. The major non‐annexin fusion activity (25% of the total cytosolic activity) was enriched by ion exchange chromatography after depletion of annexins by Ca2+‐dependent phospholipid affinity chromatography. The fusion activity co‐purified with a 10,14‐kDa dimer identified as leukocyte L1 (which was non‐fusogenic), along with an approximately 36‐kDa protein. This protein was identified as glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) by amino‐terminal sequencing, and the fusion activity was verified using commercially available GAPDH. GAPDH may play an important role in degranulation because it is as potent as annexin I on a mass basis and may constitute up to 25% of the total cytosolic fusion activity of the neutrophil. J. Leukoc. Biol. 63: 331–336; 1998.

Remodeling of Granulocyte Membrane Fatty Acids During Phagocytosis

During phagocytosis, new phospholipid is synthesized from triglyceride fatty acid and may be utilized to form the membranes of phagocytic vesicles. In addition, hydrogen peroxide, which can peroxidize unsaturated fatty acids, is generated. Since both of these processes could change membrane fatty acid composition during the conversion of cytoplasmic granules and plasma membranes to phagosomes, the lipid compositions of these structures were examined. Phagocytic vesicles were prepared by density gradient centrifugation of polystyrene latex particles after phagocytosis. Granule and plasma membrane fractions were isolated by density gradient and differential centrifugation. Phospholipids and fatty acids were analyzed by thin-layer chromatography and gas-liquid chromatography. While whole cells, granules, plasma membranes, and phagosomes were all similar in phospholipid composition, phagosome fatty acids were significantly more saturated than those of the other fractions. This was primarily due to reduced oleic and arachidonic acids and increased palmitic acid in the phagocytic vesicle lipids. Plasma membrane was also more saturated in comparison to whole cells and granules. However, this difference was not sufficient to explain the marked comparative saturation of the phagosomes. The observed increase in fatty acid saturation in these lipids may have been induced by a combination of either peroxidative destruction of polyunsaturated fatty acids or phospholipase activity, coupled with reacylation mechanisms favoring saturated fatty acids.

Human neutrophils permeabilized with digitonin respond with lysosomal enzyme release when exposed to micromolar levels of free calcium

We have recently reported that human neutrophils can be permeabilized with the cholesterol complexing agent saponin and that these cells can be induced to secrete the granule enzyme lysozyme in response to micromolar levels of free calcium. We now report that digitonin can be used in place of saponin and that it has several advantages. Permeabilization of human neutrophils was accomplished with 10 micrograms/ml digitonin in a high potassium medium. Normally impermeant solutes such as [14C]sucrose and inulin [14C]carboxylic acid gained access to one half of the intracellular water space marked with [3H]H2O. Between 30 and 100% of the cytoplasmic enzyme, lactate dehydrogenase, leaked from the intracellular space. The permeabilization process and calcium-triggered granule secretion were critically dependent upon temperature, time and digitonin concentration. Permeabilized neutrophils secreted beta-glucuronidase, lysozyme and vitamin B-12 binding-protein, constituents of both azurophil and specific granules, when exposed to micromolar levels of free calcium. Release of specific granule constituents appeared to be more sensitive to free calcium than release from azurophil granules. Although the amount of permeabilization varied considerably with each batch of cells, release of these granule markers was a consistent finding. Release of granule markers was accompanied by resealing of the cells to high-molecular-weight (Mr greater than 5000) solutes. Electron microscopic evidence also suggested that granule and plasma membranes were intact following digitonin treatment and that fusion of these membranes occurred in response to calcium. These results suggest that elevation of intracellular free-calcium levels is a sufficient condition for lysosomal enzyme release.

Role of Phorbol Diesters in In Vitro Murine Megakaryocyte Colony Formation

In vitro megakaryocyte differentiation is regulated by two activities: a megakaryocyte colony-stimulating activity (Mk-CSA), which is required for proliferation, and an auxiliary factor, megakaryocyte potentiating activity, which plays a role in later differentiation events. Tumor-promoting phorbol esters alter many cellular differentiation-related events. Thus, it was hypothesized that phorbol esters may bring about megakaryocyte differentiation in vitro. 4 beta-Phorbol 12-myristate 13-acetate (PMA), when co-cultured with a source of Mk-CSA, stimulated a threefold increase in colony numbers. Co-culture of PMA and megakaryocyte potentiator activity did not stimulate colony formation, thus eliminating any action of PMA as an Mk-CSA. The direct effect of PMA on the formation of megakaryocyte colonies was established by (a) the function of PMA as a megakaryocyte potentiator in serum-free experiments, (b) the ability of PMA to stimulate megakaryocyte colony formation using bone marrow cells depleted of populations known to produce potentiating activity, (c) the inability of bone marrow adherent cells previously treated with phorbol, 12,13-dibutyrate (PDBu) to augment megakaryocyte colony formation, and (d) the ability of PMA to induce the growth of immature megakaryocytes into large single megakaryocytes. Structure:activity experiments resulted in equivalent activities for PMA and PDBu, whereas the nontumor promoter phorbol 12,13-diacetate and phorbol itself lacked activity. The observations in this study indicate that phorbol esters can bring about megakaryocyte differentiation, and during colony formation, can induce effects identical to those brought about by biological sources of megakaryocyte potentiator activity.

Early Biochemical Events in Leukocyte Activation

The neutrophil plays a key role in host defense by ingesting and destroying foreign cells and other infectious agents. During the inflammatory process, chemotactic factors generated by bacteria, damaged tissue, the complement system and other phagocytes serve as a signaling mechanism to recruit neutrophils to sites of infection and to stimulate the cell to exercise its destructive capabilities. These responses are initiated by the interaction of activating stimuli with specific cell surface receptors, resulting in directional movement of the neutrophil toward the chemotactic source. When the neutrophil encounters an ingestible particle, its pseudopod flows around it and fuses at the distal pole of the particle, thus forming a phagosome. Once ingestion occurs, there are two major mechanisms by which the particle is eliminated. Hydrolytic and bactericidal enzymes are released from cytoplasmic granules into the phagosomal compartment by the process of degranulation. Ingested material is also vulnerable to attack by reactive oxygen metabolites produced by the phagosomal membrane. The significance of these degradative processes to host defense is best illustrated in patients with various disorders of neutrophil function who suffer from severe and recurrent infections.

PLA2 promotes fusion between PMN-specific granules and complex liposomes

Neutrophil stimulation results in the activation of a variety of phospholipases, including phospholipase A2 (PLA2), which releases arachidonic acid from the 2 position of membrane phospholipids, leaving a lysophospholipid. Because arachidonic acid is known to be a potent fusogen in vitro, we examined the effect of metabolism by PLA2 on the fusion of complex liposomes (liposomes prepared with a phospholipid composition similar to that found in neutrophil plasma membrane). We observed that PLA2 augmented the fusion of complex liposomes with each other as well as with specific granules isolated from human neutrophils, lowering the Ca2+ requirement for fusion by three orders of magnitude. Furthermore, although lysophospholipids inhibited fusion, the incorporation of arachidonic acid into liposome membranes overcame the inhibitory effects of the lysophospholipids. Thus with PLA2 and annexins we were able to obtain fusion of complex liposomes at concentations of Ca2+ that are close to physiological. Our data suggest that the activation of PLA2 and the generation of arachidonic acid may be the major fusion‐promoting event mediating neutrophil degranulation.

Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

A cell-free assay monitoring lipid mixing was used to investigate the role of Ca2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca2+ were found to directly stimulate fusion of inside-out neutrophil plasma membrane enriched fractions (from neutrophils subjected to nitrogen cavitation) with liposomes (phosphatidylethanolamine:phosphatidic acid, 4:1 molar ratio). In contrast, right-side-out plasma membranes and granule membranes did not fuse with liposomes in the presence of Ca2+. Similar results were obtained with two different lipid mixing assays. Fusion of the neutrophil plasma membrane-enriched fraction with liposomes was dependent upon the concentration of Ca2+, with threshold and 50% maximal rate of fusion occurring at 2 microM and 50 microM, respectively. Furthermore, the fusion was highly specific for Ca2+; other divalent cations such as Ba2+, Mg2+ and Sr2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca2(+)-dependent fusion did not occur between right-side-out or inside-out RBC-vesicles and liposomes. However, if the RBC-vesicles were exposed to conditions which depleted spectrin (i.e., low salt), then Ca2(+)-dependent fusion was detected. Other quantitative differences between neutrophil and RBC membranes were found; fusion of liposomes with RBC membranes was most readily achieved with La3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca2+. Furthermore, GTP gamma S was found to enhance Ca2(+)-dependent fusion between liposomes and neutrophil plasma membranes, but not RBC membranes. These studies show that plasma membranes (enriched fractions) from neutrophils are readily capable of fusing with artificial lipid membranes in the presence of micromolar concentrations of Ca2+.

The Kinetics of Secretion From Permeabilized Human Neutrophils: Release of Elastase and Correlations With Other Granule Constituents and Right Angle Light Scatter

We have previously reported that human neutrophils can be permeabilized with the cholesterol‐complexing agent digitonin. These permeabilized cells can be induced to secrete lysosomal constituents when exposed to micromolar levels of free Ca2+, a process that is enhanced by certain guanine nucleotides. We examined the kinetics in this system by employing both direct and indirect measures of secretion. A continuous, fluorescent assay of elastase permits real‐time monitoring of secretion from azurophil granules. The kinetics of elastase release proved to be rapid, beginning within 3‐10 sec and reaching a maximum at 1‐2 min. Changes in the Ca2+ concentration did not affect the “lag period” for release. A comparison of the Ca2+ dose‐response curves for release of the various granule constituents indicated that elastase was being secreted along with other contents of the azurophil granules. Changes in right angle light scatter (RLS), which have been shown to correlate closely with secretion, also commenced rapidly after the addition of Ca2+; when measured simultaneously, both the Ca2* dose‐response characteristics for changes in RLS and elastase release were very similar. Changes in RLS could be halted within 5 sec by excess EGTA and restarted promptly by repletion with secretory concentrations of Ca2+. In addition, neomycin, a phospholipase C inhibitor, profoundly diminished degranulation as monitored by RLS and end‐point techniques. A continuous assay employing 9‐aminoacridine self‐quenching as a measure of secretion proved far less satisfactory, but, nonetheless, produced similar kinetics and dose‐response characteristics. These data thus suggest that secretion of granule constituents from permeabilized neutrophils takes place rapidly and that the kinetics are comparable to those observed with intact cells.