Neutrophil Activated by the Famous and Potent PMA (Phorbol Myristate Acetate) (original) (raw)
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Phorbol ester-induced actin assembly in neutrophils: role of protein kinase C
The Journal of Cell Biology, 1992
The shape changes and membrane ruffling that accompany neutrophil activation are dependent on the assembly and reorganization of the actin cytoskeleton, the molecular basis of which remains to be clarified . A role of protein kinase C (PKC) has been postulated because neutrophil activation, with the attendant shape and membrane ruffling changes, can be initiated by phorbol esters, known activators of PKC . It has become apparent, however, that multiple isoforms of PKC with differing substrate specificities exist. To reassess the role of PKC in cytoskeletal reorganization, we compared the effects of diacylglycerol analogs and of PKC antagonists on kinase activity and on actin assembly in human neutrophils . Ruffling of the plasma membrane was assessed by scanning EM, and spatial redistribution of filamentous (F)-actin was assessed by scanning confocal microscopy. Staining with NBDphallacidin and incorporation of actin into the Triton X-100-insoluble ("cytoskeletal") fraction were used to quantify the formation of (F)-actin . [31P]ATP was used to detect protein phosphorylation in electroporated cells . Exposure of neutrophils to 4/3-PMA (an activator of PKC) induced protein phosphorylation, membrane ruffling, and assembly and reorganization of the xPOSURE of neutrophils to a variety of agents including chemotactic factors and phorbol esters initiates a coordinated series ofbiological responses . Several of these responses, including shape change, projection of membrane ruffles and pseudopodia, cell motility, and phagocytosis, are dependent on the mechanical displacement of part or all of the cell. This in turn requires the assembly and reorganization ofcytoskeletal microfilaments, which are composed primarily of actin (reviewed by . Actin also plays a major structural role in resting and activated neutrophils . Despite the importance of actin-containing microfilaments, the molecular basis of the processes that signal their assembly and reorganization
The Neutrophil: Constant Defender and First Responder
Frontiers in Immunology, 2020
The role of polymorphonuclear neutrophils (PMNs) in biology is often recognized during pathogenesis associated with PMN hyper-or hypo-functionality in various disease states. However, in the vast majority of cases, PMNs contribute to resilience and tissue homeostasis, with continuous PMN-mediated actions required for the maintenance of health, particularly in mucosal tissues. PMNs are extraordinarily well-adapted to respond to and diminish the damaging effects of a vast repertoire of infectious agents and injurious processes that are encountered throughout life. The commensal biofilm, a symbiotic polymicrobial ecosystem that lines the mucosal surfaces, is the first line of defense against pathogenic strains that might otherwise dominate, and is therefore of critical importance for health. PMNs regularly interact with the commensal flora at the mucosal tissues in health and limit their growth without developing an overt inflammatory reaction to them. These PMNs exhibit what is called a para-inflammatory phenotype, and have reduced inflammatory output. When biofilm growth and makeup are disrupted (i.e., dysbiosis), clinical symptoms associated with acute and chronic inflammatory responses to these changes may include pain, erythema and swelling. However, in most cases, these responses indicate that the immune system is functioning properly to re-establish homeostasis and protect the status quo. Defects in this healthy everyday function occur as a result of PMN subversion by pathological microbial strains, genetic defects or crosstalk with other chronic inflammatory conditions, including cancer and rheumatic disease, and this can provide some avenues for therapeutic targeting of PMN function. In other cases, targeting PMN functions could worsen the disease state. Certain PMN-mediated responses to pathogens, for example Neutrophil Extracellular Traps (NETs), might lead to undesirable symptoms such as pain or swelling and tissue damage/fibrosis. Despite collateral damage, these PMN responses limit pathogen dissemination and more severe damage that would otherwise occur. New data suggests the existence of unique PMN subsets, commonly associated with functional diversification in response to particular inflammatory challenges. PMN-directed therapeutic approaches depend on a greater understanding of this diversity. Here we outline the current understanding of PMNs in health and disease, with an emphasis on the positive manifestations of tissue and organ-protective PMN-mediated inflammation.
Plasticity of neutrophils reveals modulatory capacity
Brazilian Journal of Medical and Biological Research, 2015
Neutrophils are widely known as proinflammatory cells associated with tissue damage and for their early arrival at sites of infection, where they exert their phagocytic activity, release their granule contents, and subsequently die. However, this view has been challenged by emerging evidence that neutrophils have other activities and are not so short-lived. Following activation, neutrophil effector functions include production and release of granule contents, reactive oxygen species (ROS), and neutrophil extracellular traps (NETs). Neutrophils have also been shown to produce a wide range of cytokines that have pro-or anti-inflammatory activity, adding a modulatory role for this cell, previously known as a suicide effector. The presence of cytokines almost always implies intercellular modulation, potentially unmasking interactions of neutrophils with other immune cells. In fact, neutrophils have been found to help B cells and to modulate dendritic cell (DC), macrophage, and T-cell activities. In this review, we describe some ways in which neutrophils influence the inflammatory environment in infection, cancer, and autoimmunity, regulating both innate and adaptive immune responses. These cells can switch phenotypes and exert functions beyond cytotoxicity against invading pathogens, extending the view of neutrophils beyond suicide effectors to include functions as regulatory and suppressor cells.
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
Chemotactic Signaling Pathways in Neutrophils: from Receptor to Actin Assembly
Critical Reviews in Oral Biology & Medicine, 2002
In this review, we present an overview of the signaling elements between neutrophil chemotactic receptors and the actin cytoskeleton that drives cell motility. From receptor-ligand interactions, activation of heterotrimeric G-proteins, their downstream effectors PLC and PI-3 kinase, the activation of small GTPases of the Rho family, and their regulation of particular cytoskeletal regulatory proteins, we describe pathways specific to the chemotaxing neutrophil and elements documented to be important for neutrophil function.
The Journal of Immunology
The generation of superoxide anion and release of granule contents are essential to the bactericidal function of neutrophils, but may also contribute to host tissue damage during inflammation. In previous studies (J. Immunol. 146:2388), we have demonstrated that the acute phase reactant alpha-1-antichymotrypsin (ACT), a potent inhibitor of the serine protease cathepsin G, also suppresses superoxide anion generation. The inhibitory effect of ACT was not directly linked to its antiproteolytic activity and may reflect interaction at a site other than its reactive loop. To further characterize the mechanism of inhibition, we investigated the direct effects of ACT on the NADPH oxidase enzyme complex and the signaling pathways that regulate motivation of the respiratory burst. We present evidence that ACT does not intefer with agonist-stimulated calcium mobilization or translocation and activity of protein kinase C. ACT was an effective inhibitor of superoxide anion generation in membrane...
Diverse stimuli engage different neutrophil extracellular trap pathways
eLife, 2017
Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways induced by five stimuli; PMA, the calcium ionophore A23187, nigericin, Candida albicans and Group B Streptococcus. We studied NET production in neutrophils from healthy donors with inhibitors of molecules crucial to PMA induced NETs including protein kinase C, calcium, reactive oxygen species, the enzymes myeloperoxidase (MPO) and neutrophil elastase. Additionally, neutrophils from chronic granulomatous disease patients, carrying mutations in the NADPH oxidase complex or a MPO-deficient patient were examined. We show that PMA, C. albicans and GBS use a related pathway for NET induction whereas ionophores require an alternative pathway but that NETs produced by all stimuli are proteolytically active, kill bacteria and composed mainly of chromosomal DNA. Thus, we demonstrate that NETosis occurs through several signalling ...
Two Signaling Mechanisms for Activation of alpha Mbeta 2 Avidity in Polymorphonuclear Neutrophils
Journal of Biological Chemistry, 1998
Circulating polymorphonuclear neutrophils (PMN) are quiescent, nonadherent cells that rapidly activate at sites of inflammation, where they develop the capacity to perform a repertoire of functions that are essential for host defense. Induction of integrin-mediated adhesion, which requires an increase in integrin avidity, is critical for the development of these effector functions. Although a variety of stimuli can activate integrins in PMN, the signaling cascades involved are unclear. Phosphatidylinositol (PI) 3-kinase has been implicated in integrin activation in a variety of cells, including PMN. In this work, we have examined activation of the PMN integrin ␣ M  2 , assessing both adhesion and generation of the epitope recognized by the activation-specific antibody CBRM1/5. We have found that PI 3-kinase has a role in activation of ␣ M  2 by immune complexes, but we have found no role for it in ␣ M  2 activation by ligands for trimeric G protein-coupled receptors, including formylmethionylleucylphenylalanine (fMLP), interleukin-8, and C5a. Cytochalasin D inhibition suggests a role for the actin cytoskeleton in immune complex activation of ␣ M  2 , but cytochalasin has no effect on fMLP-induced activation. Similarly, immune complex activation of the Rac/Cdc42-dependent serine/threonine kinase Pak1 is blocked by PI 3-kinase inhibitors, but fMLP-induced activation is not. These results demonstrate that two signaling pathways exist in PMN for activation of ␣ M  2 . One, induced by Fc␥R ligation, is PI 3-kinase-dependent and requires the actin cytoskeleton. The second, initiated by G protein-linked receptors, is PI 3-kinase-independent and cytochalasin-insensitive. Pak1 may be in a final common pathway leading to activation of ␣ M  2 .
PloS one, 2013
Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK) downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP), and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca 2+ signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.