LOCALIZED IN SPECIFIC GRANULES AND IS TRANSLOCATED TO THE CELL SURFACE BY EXOCYTOSIS (original) (raw)
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Regulation of plasminogen binding to neutrophils
Blood, 2001
Plasminogen plays an integral role in the inflammatory response, and this participation is likely to depend on its interaction with cell surfaces. It has previously been reported that isolation of human neutrophils from blood leads to a spontaneous increase in their plasminogen-binding capacity, and the basis for this up-regulation has been explored as a model for mechanisms for modulation of plasminogen receptor expression. Freshly isolated human peripheral blood neutrophils exhibited relatively low plasminogen binding, but when cultured for 20 hours, they increased this capacity dramatically, up to 50-fold. This increase was abolished by soybean trypsin inhibitor and was susceptible to carboxypeptidase B treatment, implicating proteolysis and exposure of carboxy-terminal lysines in the enhanced interaction. In support of this hypothesis, treatment of neutrophils with elastase, cathepsin G, or plasmin increased their plasminogen binding, and specific inhibitors of elastase and cath...
Journal of Experimental Medicine, 1996
The pathogenic role of antineutrophil cytoplasmic autoantibodies (ANCA) remains controversial because of the difficulty in explaining how extracellular ANCA can interact with intracellular primary granule constituents. It has been postulated that cytokine priming of neutrophils (PMN), as may occur during a prodromal infection, is an important trigger for mobilization of granules to the cell surface, where they may interact with ANCA. We show by electron microscopy that apoptosis of unprimed PMN is also associated with the translocation of cytoplasmic granules to the cell surface and alignment just beneath an intact cell membrane. Immunofluorescent microscopy and FACS ® analysis demonstrate reactivity of ANCA-positive sera and antimyeloperoxidase antibodies with apoptotic PMN, but not with viable PMN. Moreover, we show that apoptotic PMN may be divided into two subsets, based on the presence or absence of granular translocation, and that surface immunogold labeling of myeloperoxidase occurs only in the subset of PMN showing translocation. These results provide a novel mechanism that is independent of priming, by which ANCA may gain access to PMN granule components during ANCA-associated vasculitis. A ntineutrophil cytoplasmic autoantibodies (ANCA) 1 are associated with systemic vasculitides, especially Wegener's granulomatosis and microscopic polyarteritis (1-4). ANCA are also seen with idiopathic crescentic glomerulonephritis without immune deposits (2), and several other inflammatory or rheumatic diseases (3, 4). These autoAb are mainly directed against proteins in PMN primary granules and monocyte lysosomes (5). When detected by indirect immunofluorescence (IF) of ethanol-fixed PMN, there are two major patterns of ANCA staining-cytoplasmic (C-ANCA) and perinuclear (P-ANCA) (2). The major C-ANCA Ag is proteinase 3 (PR3) (6), a 29 kD serine proteinase. The major P-ANCA Ag is myeloperoxidase (MPO) (2). Although PR3 and MPO are located in the primary granules of PMN, ethanol fixation leads to solubilization and nuclear redistribution of MPO, leading to an artifactual perinuclear staining pattern (2, 7). Other minor ANCA Ag have been described, leading to both C-and P-ANCA patterns, but these account for Ͻ 5% of positive ANCA (5). The pathogenic role of ANCA remains controversial, in part because it is difficult to explain how extracellular ANCA interact with intracellular primary granule components. Al
The Journal of Cell Biology
Neutrophils stimulated by the chemotactic factor formyl-methionyl-leucyl-phenylalanine (FMLP) undergo a transient change in surface properties that permits the cells to adhere more readily to surfaces and to each other. This transient change can be monitored by light scattering as stimulated neutrophils form aggregates while stirred in a platelet aggregometer. Maximum change in light scattering occurs within 1 min and correlates with an increase in the percentage of cells that are in aggregates of four or more cells and a decrease in the percentage of single cells. With time (3-5 min), small aggregates disappear and single cells reappear. The transient change in adhesiveness is accompanied by a persistent change in cell shape; the cells become polarized and protrude ruffles from one sector of the cell surface. During aggregation the cells adhere to one another with smooth sides together and ruffles pointed outward. During disaggregation the cells dissociate laterally with the simultaneous internalization of membrane in the region opposite the ruffles. Particle bound to the surface by charge (thorotrast, cationized ferritin) are concentrated and internalized in this region. The change in cell shape from round to ruffled occurs within seconds, suggesting that membrane is added to the cell surface from an intracellular store. We therefore quantified surface membrane by electron microscopy morphometry and measured a 25% increase within 10 s of adding FMLP. The source of new membrane appeared to be the specific granule membrane since the kinetics of granule discharge (between 30% and 50% of all release occurs in the first 10 s) correlate with the appearance of new membrane. Furthermore, the amount of membrane that appears at the cell surface at 10 s correlates with that lost from intracellular granules in that time. Chemotaxin-induced aggregation thus begins with granule discharge and membrane addition followed by protrusion of ruffles. Adherence is maximal at 60 s and the gradual loss of adhesiveness that follows is associated with uropod formation and enhanced endocytic activity.
The urokinase-plasminogen activator system in ovine macrophages and neutrophils
Small Ruminant Research, 2002
The urokinase-plasminogen activator (u-PA) system in resting and activated ovine macrophages and neutrophils was examined. Macrophages and neutrophils were isolated from a total of 28 lactating sheep of the Chios breed. Low amounts of u-PA were found intracellularly or membrane-bound in resting macrophages and neutrophils. However, incubation of resting macrophages or neutrophils with purified u-PA (10 IU/ml) revealed extensive binding of u-PA to cell membranes. Excess amino terminal fragment of the u-PA molecule, a proteolytically inactive fragment of u-PA (amino acids 1-135) blocked binding of u-PA to macrophage or neutrophil cell membrane. These results indicate that the binding of u-PA is specific and that resting neutrophils and macrophages have unoccupied u-PA receptors on their cell membrane. Addition of phorbol myristate acetate (PMA) led to an increase ðP < 0:01Þ in total cell-associated and membrane-bound u-PA activity and a decrease ðP < 0:01Þ in free, unoccupied u-PA binding sites of macrophages or neutrophils. No significant effects on total cellassociated or membrane-bound u-PA were found when macrophages or neutrophils were treated with 4-phorbol-12,13didecanoate, a phorbol ester that does not activate protein kinase C (PKC). Furthermore, the PMA-induced increase in total cell-associated u-PA activity of sheep macrophages or neutrophils was completely ablated by the PKC inhibitor 1-(5isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 100 mM) but was unaffected by the cyclic nucleotidedependent protein kinase inhibitor N-(2-quanidinoethyl)-5-isoquinoline sulfonamide hydrochloride (HA-1004, 100 mM). Thus, PKC plays a role in the modulation of u-PA system by PMA in ovine macrophages and neutrophils.
Urokinase-Type Plasminogen Activator Inhibits Efferocytosis of Neutrophils
American Journal of Respiratory and Critical Care Medicine, 2010
The urokinase-type plasminogen activator (u-PA)/plasmin system plays an important role in promoting cell migration and invasion, an effect which is largely ascribed to the proteolytic activity of these enzymes. We investigated whether u-PA modulates integrin-dependent T lymphocyte migration and adhesion on fibronectin independently of its plasminogen activator function. Here we report that u-PA reduced the spontaneous and phorbol 12-myristate 13-acetate-induced migration of peripheral blood T lymphocytes on fibronectin by 20-50 %, decreased the T lymphocyte and § 4 g 1 + / § 5 g 1 + K562 cell adhesion on fibronectin by 30-40 %, and completely suppressed integrin § 4 g 1-dependent T lymphocyte and § 4 g 1 + / § 5 g 1 + K562 cell adhesion to the LDV-containing 40-kDa fibronectin fragment. The u-PA receptor was not essential for this effect. In contrast, adhesion of § 4 g 1 -/ § 5 g 1 + K562 cells to an RGD-containing fibronectin fragment was unaffected. A recombinant protein comprising the N-terminal fragment of u-PA, but lacking its proteolytic domain, had the same inhibitory effect. Decreased adhesion was neither associated with a diminished cell surface expression of § 4 g 1 nor with a suppression of § 4 g 1 ligand-binding function. Our results demonstrate that u-PA inhibits § 4 g 1-but not § 5 g 1-mediated lymphocyte/leukocyte adhesion to fibronectin independently of its proteolytic activity. This finding provides additional evidence that matrix proteinases may participate in cell adhesion and migration control independently of their matrix-degrading activity. Abbreviations: ATF: Amino-terminal fragment ECM: Extracellular matrix FN: Fibronectin fnIII: Fibronectin type III repeat LIBS: Ligand-induced binding site MMP: Matrix metalloproteinase u-PA: Urokinase-type plasminogen activator u-PAR: u-PA receptor P. Olivier et al.
FEBS Letters, 1992
In order to identify cytosolic proteins involved in control of granule esocytosis in human neutrophils, subcellular fractions enriched in each of the 3 major granule subsets were incubated with cytosol l'rom neutrophils in the presence or absence of Ca:'. After washing, proteins were eluted from the organelles by EGTA, Annexins I, 11, IV and VI were found to bind to all organelles studied. In addition, a 28-kDa protein was found to bind exclusively to plasma membranes and secretory vesicles, the most readily ¢xocytosed organelle of neutrophils. CaZ'.-dependent association ,r cytosolic proteins to different granule subsets may control differential exoeytosis of granules.
Sequential Chemotactic and Phagocytic Activation of Human Polymorphonuclear Neutrophils
Infection and Immunity, 2007
Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants' origins are reached, specific receptors bind to the invader's surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (⌬[Ca 2؉ ] i and ⌬pH i ) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucylphenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca 2؉ ] i , mostly from intracellular stores, simultaneously with a drop in pH i ; these are followed by a drop in [Ca 2؉ ] i and a rise in pH i , with the latter being due to a Na ؉ /H ؉ antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca 2؉ ] i is restored, 10 ؊7 M fMLP, previously shown to elicit maximal ⌬[Ca 2؉ ] i but no bactericidal functions, did not prevent the cells' responses with ⌬[Ca 2؉ ] i to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 g/ml IC, previously shown to elicit maximal ⌬[Ca 2؉ ] i and bactericidal functions, exhibited no subsequent ⌬[Ca 2؉ ] i or ⌬pH i to either stimulus. While exposure to 10 ؊7 M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10 ؊5 M fMLP did, presumably via the low-affinity receptor, using a different Ca 2؉ source. on February 9, 2015 by guest http://iai.asm.org/ Downloaded from FIG. 4. Sequential stimulation of PMN by saturating doses (120 g/ml) of IC followed 5 min later by 10 Ϫ7 M fMLP. (a) Control; (b) 5 mM EGTA added 15 s before IC injection; (c) 5 mM EGTA added before fMLP injection; (d) release of elastase (shown as F 460 ) and of oxidative products (shown as F 530 ). Each figure is representative of five independent experiments. VOL. 75, 2007 SEQUENTIAL CHEMOTACTIC AND IC ACTION ON PMN 3995 on February 9, 2015 by guest
Neutrophil Activated by the Famous and Potent PMA (Phorbol Myristate Acetate)
Cells
This review will briefly outline the major signaling pathways in PMA-activated neutrophils. PMA is widely used to understand neutrophil pathways and formation of NETs. PMA activates PKC; however, we highlight some isoforms that contribute to specific functions. PKC α, β and δ contribute to ROS production while PKC βII and PKC ζ are involved in cytoskeleton remodeling. Actin polymerization is important for the chemotaxis of neutrophils and its remodeling is connected to ROS balance. We suggest that, although ROS and production of NETs are usually observed together in PMA-activated neutrophils, there might be a regulatory mechanism balancing both. Interestingly, we suggest that serine proteases might determine the PAD4 action. PAD4 could be responsible for the activation of the NF-κB pathway that leads to IL-1β release, triggering the cleavage of gasdermin D by serine proteases such as elastase, leading to pore formation contributing to release of NETs. On the other hand, when serine ...