Browsing by Subject "Macrófagos"

Now showing 1 - 8 of 8
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    Apoptosis-associated speck-like protein containing CARD forms specks but does not activate caspase-1 in the absence of NLRP3 during macrophage swelling
    (The American Association of Immunologists, Inc., 2015) Compan, Vincent; Martín-Sánchez, Fátima; Baroja-Mazo, Alberto; López-Castejón, Gloria; Gomez, Ana I.; Verkhratsky, Alexei; Brough, David; Pelegrin Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
    Apoptosis-associated speck-like protein containing a CARD (ASC) is a key adaptor molecule required for inflammatory processes. ASC acts by bridging NLRP proteins, such as NLRP3, with pro-caspase-1 within the inflammasome complex that subsequently results in the activation of caspase-1 and the secretion of interleukin (IL)-1b and IL-18. In response to bacterial infection, ASC also forms specks by self-oligomerization to activate caspase-1 and induce pyroptosis. Hitherto the role of these specks in NLRP3 inflammasome activation in response to danger signals is largely unexplored. Here we report that under hypotonic conditions, ASC formed specks independently of NLRP3 that did not activate caspase-1. These specks were not associated with pyroptosis and were controlled by Transient Receptor Potential Vanilloid 2 channel mediated signaling. However, interaction with NLRP3 enhanced ASC speck formation leading to fully functional inflammasomes and caspase-1 activation. This study reveals that the ASC speck could present different oligomerization assemblies and represents an essential step in the activation of functional NLRP3 inflammasomes.
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    Dynamics of macrophage polarization reveal new mechanism to inhibit IL-1beta release through pyrophosphates
    (EMBO Press, 2009) Pelegrin, Pablo; Surprenant, Annmarie; Bioquímica y Biología Molecular B e Inmunología
    In acute inflammation extracellular ATP activates P2X7 ion channel receptors (P2X7R) on M1 polarized macrophages to release pro-inflammatory IL-1bvia activation of the caspase-1/Nucleotide-binding domain and Leucine-rich repeat receptor containing Pyrin domain 3 (NLRP3) inflammasome. In contrast, M2 polarized macrophages are critical to the resolution of inflammation but neither actions of P2X7R on these macrophages, nor mechanisms by which macrophages switch from pro-inflammatory to anti-inflammatory phenotypes, are known. Here we investigated extracellular ATP signaling over a dynamic macrophage polarity gradient from M1 through M2 phenotypes. In macrophages polarized towards, but not at, M2 phenotype, where intracellular IL-1b remains high and the inflammasome is intact, P2X7R activation selectively uncouples to the NLRP3-inflammasome activation but not to upstream ion channel activation. In these intermediate M1/M2 polarized macrophages, extracellular ATP now acts via its pyrophosphate chains, independently of other purine receptors, to inhibit IL-1b release by other stimuli via two independent mechanisms: inhibition of ROS production and trapping of the inflammasome complex via intracellular clustering of actin filaments.
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    Extracellular ATP activates the NLRP3 inflammasome and is an early danger signal of skin allograft rejection
    (Cell Press, 2017) Amores-Iniesta, Joaquín; Barberá-Cremades, Maria; Martínez, Carlos M.; Pons, José A.; Revilla-Nuín, Beatriz; Martínez-Alarcón, Laura; Di Virgilio, Francesco; Parrilla, Pascual; Baroja-Mazo, Alberto; Pelegrín, Pablo; Bioquímica y Biología Molecular B e Inmunología
    Immune cells are equipped with a number of receptors that recognize sterile injury and pathogens. We find that host immune cells release ATP as an inflammatory signal in response to allogeneic transplantation. ATP then acts via a feedback mechanism on the P2X7 channel to activate the NLRP3 inflammasome and subsequently process and release interleukin (IL)-18. This process is a necessary stage in the deleterious Th1 response against allotransplantation via interferon-g production. Lack of IL-18 resulted in a decrease in graft- infiltrated CD8 cells, but an increase in regulatory T cells. In human liver transplant patients subjected to progressive immunosuppressive drug withdrawal, we found that patients suffering acute rejection had higher levels of the P2X7 receptor in circulating inflammatory monocytes compared to tolerant patients. These data suggest that the pharmacological inhibition of the P2X7 receptor or the NLRP3 inflammasome will aid in inducing transplant tolerance without complete immunoparalysis.
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    Inflammasome-dependent IL-1β release depends upon membrane permeabilisation
    (Nature, 2016) Martín-Sáncez, Fátima; Diamon, Catherine; Zeitler, Marcel; Gomez-Sanchez, Ana; Baroja-Mazo, Alberto; Bagnall, James; Spiller, David; White, Michael; Daniels, Michael J. D.; Mortellaro, Alessandra; Peñalver, Marcos; Paszek, Pawel; Steringer, Julia P.; Nickel, Walter; Brough, David; Pelegrin Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
    Interleukin (IL)-1β is a critical regulator of the inflammatory response. IL-1β is not secreted through the conventional ER-Golgi route of protein secretion and to-date its mechanism of release has been unknown. Crucially its secretion depends upon the processing of a precursor form following the activation of the multi-molecular inflammasome complex. Using a novel and reversible pharmacological inhibitor of the IL-1β release process, in combination with biochemical, biophysical and real-time single-cell confocal microscopy with macrophage cells expressing Venus labelled IL-1β, we have discovered that the secretion of IL-1β after inflammasome activation requires membrane permeabilisation, and occurs in parallel with the death of the secreting cell. Thus in macrophages the release of IL-1β in response to inflammasome activation appears to be a secretory process independent of non-specific leakage of proteins during cell death. The mechanism of membrane permeabilisation leading to IL-1β release is distinct from the unconventional secretory mechanism employed by its structural homologues FGF2 or IL-1α, a process that involves the formation of membrane pores but does not result in cell death. These discoveries reveal key processes at the initiation of an inflammatory response and deliver new insights into mechanisms of protein release.
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    Interleukin-1β isolated from a marine fish reveals up-regulated expression in macrophages following activation with lipopolysaccharide and lymphokines
    (Elsevier, 2001) Pelegrin, Pablo; García-Castillo, Jesús; Mulero, Victoriano; Meseguer, José; Bioquímica y Biología Molecular B e Inmunología
    The gilthead seabream IL-1β gene consists of five exons/four introns. The complete coding sequence contains a 102 bp 5’ untraslated region (UTR), a single open reading frame of a 762 bp which translates into a 253 amino acid molecule, and a 407 bp 3’UTR with a polyadenilation signal 14 nucleotides upstream of the poly(A)tail. The seabream sequence has highest degree of nucleotide (61.7%) and amino acid (53%) identity with the trout IL-1β sequences. The IL-1β message was detected by RT-PCR in head-kidney, blood, spleen, liver, gill and peritoneal exudate of both non-infected and Vibrio anguillarum-challenged fish. More importantly, IL-1β was highly expressed by purified macrophage monolayers and was up-regulated by lipopolysaccharide and lymphocyte-derived macrophage-activating factor stimulation.
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    P2X7 receptor differentially couples to distinct release pathways for IL-1b in mouse macrophage
    (The American Association of Immunologists, Inc., 2008) Pelegrin, Pablo; Barroso-Gutierrez, Consuelo; Surprenant, Annmarie; Bioquímica y Biología Molecular B e Inmunología
    The pro-inflammatory IL-1 cytokines, IL-1a, IL-1b and IL-18, are key mediators of the acute immune response to injury and infection. Mechanisms underlying their cellular release remain unclear. Activation of purinergic P2X7 receptors (P2X7R) by extracellular ATP is a key physiological inducer of rapid IL-1brelease from LPS-primed macrophage. We investigated patterns of ATP-mediated release of IL-1 cytokines from three macrophage types in attempts to provide direct evidence for or against distinct release mechanisms. We used peritoneal macrophage from P2X7R-/- mice and found that release of IL-1a, IL-18, as well as IL-1b, by ATP resulted exclusively from activation of P2X7R, that release of all these IL-1 cytokines involved pannexin-1 (panx1), and that there was both a panx1-dependent and independent component to IL-1b release. We compared IL-1 release patterns from LPS-primed peritoneal macrophage, RAW264.7 macrophage and J774A.1 macrophage. We found RAW264.7 macrophage readily release pro-IL- 1b independently of panx1 but do not release mature IL-1b because they do not express apoptotic speck-like protein with a caspase-activating recruiting domain (ASC) and so have no caspase-1 inflammasome activity. We delineated two distinct release pathways: the well-known caspase-1 cascade mediating release of processed IL-1b that was selectively blocked by inhibition of caspase-1 or panx1, and a calcium-independent, caspase-1/panx1-independent release of pro-IL-1b that was selectively blocked by glycine. None of these release responses were associated with cell damage or cytolytic effects. This provides the first direct demonstration of a distinct signaling mechanism responsible for ATP-induced release of pro-IL-1b.
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    Pannexin-1 couples to maitotoxin and nigericin- induced il-1β release through a dye-uptake independent pathway
    (Elsevier, 2007) Pelegrin, Pablo; Surprenant, Annmarie; Bioquímica y Biología Molecular B e Inmunología
    Pannexin-1 is a recently identified membrane protein that can act as a non-selective pore permeable to dyes such as ethidium when ectopically expressed. Blockade of pannexin-1 in macrophage endogenously expressing the ATP-gated P2X7 receptor (P2X7R) blocks the initial dye uptake, but not the ionic current, and also blocks processing and release of interleukin-1β (IL-1β) in response to P2X7R activation. These results suggest that pannexin-1 may be a hemichannel activated by the P2X7R to provide the conduit for dye uptake and downstream signalling to processing and release of IL-1β. We have pursued this hypothesis by measuring dye uptake and IL-1β processing and release in mouse J774 macrophage in response to P2X7R activation and to maitotoxin and nigericin, two agents considered to evoke IL-1β release via the same mechanism. Experiments were carried out over time periods during which no LDH was released from cells in order to examine only non-cytolytic pathways. P2X7R activation evoked dye-uptake that could be separated into two components by pannexin-1 inhibition: an initial rapid phase and a slower pannexin-1- independent phase. Maitotoxin evoked dye uptake was unaltered by pannexin-1 inhibition. Nigericin did not induce dye uptake. Inhibition of pannexin-1 blocked caspase-1 and IL-1β processing and release in response to all three stimuli. Thus, while pannexin-1 is required for IL-1β release in response to maitotoxin, nigericin and ATP, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes.
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    The NLRP3 inflammasome is released as a particulate danger signal that amplifies the inflammatory response
    (Nature, 2014) Baroja-Mazo, Alberto; Martín-Sánchez, Fátima; Gomez, Ana I.; Martínez, Carlos M.; Amores-Iniesta, Joaquín; Compan, Vincent; Barberá-Cremades, María; Yagüe, Jordi; Ruiz-Ortiz, Estibaliz; Antón, Jordi; Buján, Segundo; Couillin, Isabelle; Brough, David; Arostegui, Juan I.; Pelegrin Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
    NLRP3 inflammasome assembly activates caspase-1 and mediates the processing and release of the leaderless cytokine IL-1β, and thereby plays a central role in the inflammatory response and in diverse human diseases. Here we report that upon caspase-1 activation oligomerized NLRP3-inflammasome particles are released from macrophages. Recombinant oligomeric protein particles composed of the adapter protein ASC or the cryopyrin-associated periodic syndromes (CAPS) mutant NLRP3 p.D303N, stimulate further caspase-1 activation extracellularly, and also intracellularly upon phagocytosis by surrounding macrophages. ASC oligomeric particles were found in the serum of patients with active CAPS, but not in patients with other inherited autoinflammatory diseases. Our findings support a model whereby the NLRP3-inflammasome, acting as an extracellular oligomeric complex, amplifies the inflammatory response.