DigitalUM :: Browsing by Subject "Citocinas"

Browsing by Subject "Citocinas"

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Open Access
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.
Open Access
Cell volume regulation modulates NLRP3 inflammasome activation
(Cell Press, 2012) Compan, Vincent; Baroja-Mazo, Alberto; Lopez-Castejón, Gloria; Gomez, Ana I.; Martínez, Carlos M.; Angosto, Diego; Montero, María T.; Herranz, Antonio S.; Bazán, Eulalia; Reimers, Diana; Mulero, Victoriano; Pelegrín Vivancos, Pablo; Bioquímica y Biología Molecular B e Inmunología
Cell volume regulation is a primitive response to alterations in environmental osmolarity. The NLRP3 inflammasome is a multiprotein complex that senses pathogen- and danger-associated signals. Here we report that the basic mechanisms of cell swelling and regulatory volume decrease (RVD) are sensed from fish to mammals by the NLRP3 inflammasome. We found that a decrease in extracellular osmolarity induced (i) a K+-dependent conformational change of the preassembled NLRP3-inactive inflammasome during cell swelling, followed by (ii) activation of the NLRP3 inflammasome and caspase-1, which was controlled by Transient Receptor Potential (TRP) channels during RVD. Both mechanisms were necessary for interleukin-1b processing. Increased extracellular osmolarity prevented caspase-1 activation by different known NLRP3 activators. Collectively, our data place cell volume regulation as a basic conserved homeostatic mechanism associated with the formation of the NLRP3 inflammasome and provides a mechanism for NLRP3 inflammasome activation.
Open Access
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.
Open Access
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.
Embargo
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.
Open Access
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.
Open Access
Pannexin-1 mediates large pore formation and interleukin-1ß release by the ATP-gated P2X7 receptor
(2006) Pelegrin, Pablo; Surprenant, Annmarie; Bioquímica y Biología Molecular B e Inmunología
P2X(7) receptors are ATP-gated cation channels; their activation in macrophage also leads to rapid opening of a membrane pore permeable to dyes such as ethidium, and to release of the pro-inflammatory cytokine, interleukin-1beta (IL-1beta). It has not been known what this dye-uptake path is, or whether it is involved in downstream signalling to IL-1beta release. Here, we identify pannexin-1, a recently described mammalian protein that functions as a hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin-1 is required for processing of caspase-1 and release of mature IL-1beta induced by P2X(7) receptor activation.
Open Access
Performance validation of quantitative polymerase chain reaction assays for measuring swine proinflammatory and immunomodulatory cytokine gene expression
(Universidad de Murcia, 2015) García-Nicolás, Obdulio; Juan José Quereda; Jaime Gómez-Laguna; Irene Magdalena Rodríguez-Gómez; Librado Carrasco; Guillermo Ramis; Francisco José Pallarés
ABSTRACT: RT-qPCR is the method of choice for the accurate detection of low quantities of mRNA due to its higher sensitivity and specificity. Most of the previously published reports about swine cytokine gene expression lack information regarding the validation of the technique, which impedes the potential implementation by new users. This study was focussed on the technical validation of already published RT-qPCR assays for swine proinflammatory (IFN-α, IFN-γ, IL-12p35, IL-12p40 and TNF-α) and immunomodulatory (IL-10 and TGF-β) cytokines, and on defining the best qPCR amplification conditions for simultaneous amplification of the selected cytokines from several porcine tissue samples. The tested RT-qPCR assays here are sensitive (Efficient close to 2, Correlation coefficient higher than 0.95 and a Limit Of Detection below 305-100 mRNA copies), robust (Coefficint of Variation and Factor of Discrimination means were lower than 5 and 3%, respectively) and highly useful for the study of immune swine responses.
Open Access
Phylogeny of cytokines: molecular cloning and expression analysis of sea bass Dicentrarchus labrax interleukin-1b
(2001) Scapigliati, G.; Buonocore, F.; Bird, S.; Zou, J.; Pelegrín, Pablo; Falasca, C.; Prugnoli, D.; Secombes, C. J.; Bioquímica y Biología Molecular B e Inmunología
In this paper the cloning of interleukin-1b (IL-1b) from the fish Dicentrarchus labrax (sea bass) is described. Using degenerate primers designed from known IL-1b sequences, a cDNA fragment was amplified by PCR and elongated by 3’ and 5’ RACE to give the full-length coding sequence for sea bass IL-1. The cDNA is 1292 bp, lacks a putative ICE cut site, and codes for a deduced peptide of 29.4 kDa with a pI of 5.1. Sequence analysis showed highest amino acid similarity with rainbow trout (62%), Xenopus (46%), and carp (45.5%) IL-1b sequences. Expression studies show that sea bass IL-1b can be upregulated by bacterial lipopolysaccharide both in vitro and in vivo in leucocytes from blood, head-kidney, spleen, gills and liver, whereas the IL-1b transcript was not detectable in thymus and gut-associated lymphoid tissue. Northern blot analysis with head-kidney leucocyte RNA showed a main LPS- upregulated band at 1.3 kb, and two minor bands at 0.9 and 3.0 kb, respectively. Phylogenetic comparisons with IL-1b from other vertebrates is presented.
Open Access
Production and mechanism of secretion of interleukin-1b from the marine fish gilthead seabream
(2004) Pablo, Pelegrin; Elena, Chaves-Pozo; José, Meseguer; Victoriano, Mulero; Bioquímica y Biología Molecular B e Inmunología
Interleukin-1b (IL-1b) is a secretory cytokine lacking a signal peptide, and does not follow the classical endoplasmic reticulum to Golgi pathway of secretion. Its post-translational processing by IL-1b-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors. No information is available on the production and release of fish IL-1b, but the IL-1b gene sequences reported to date lack a conserved ICE recognition site. We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor (MAF)/bacterial DNA (VaDNA)-primed immune cells of fish accumulate intracellular IL-1b as a ~30 kDa polypeptide (proIL-1b). The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor (PRR). More importantly, addition of extracellular ATP does not promote IL-1b secretion by immune cells and fails to induce phosphatidylserine (PS) flip. In contrast, fish SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1b form within 30 min of activation with ATP. Notably, the post-translational processing of IL-1b by SAF-1 cells is abrogated by a specific ICE inhibitor.
Open Access
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.

Browsing by Subject "Citocinas"

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