Dynamics of macrophage polarization reveal new mechanism to inhibit IL-1beta release through pyrophosphates.

In acute inflammation, extracellular ATP activates P2X(7) ion channel receptors (P2X(7)R) on M1 polarized macrophages to release pro-inflammatory IL-1beta through 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 P2X(7)R on these macrophages nor mechanisms by which macrophages switch from pro-inflammatory to anti-inflammatory phenotypes are known. Here, we investigated extracellular ATP signalling over a dynamic macrophage polarity gradient from M1 through M2 phenotypes. In macrophages polarized towards, but not at, M2 phenotype, in which intracellular IL-1beta remains high and the inflammasome is intact, P2X(7)R 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 through its pyrophosphate chains, independently of other purine receptors, to inhibit IL-1beta release by other stimuli through two independent mechanisms: inhibition of ROS production and trapping of the inflammasome complex through intracellular clustering of actin filaments.

Signaling at purinergic P2X receptors.

P2X receptors are membrane cation channels gated by extracellular ATP. Seven P2X receptor subunits (P2X(1-7)) are widely distributed in excitable and nonexcitable cells of vertebrates. They play key roles in inter alia afferent signaling (including pain), regulation of renal blood flow, vascular endothelium, and inflammatory responses. We summarize the evidence for these and other roles, emphasizing experimental work with selective receptor antagonists or with knockout mice. The receptors are trimeric membrane proteins: Studies of the biophysical properties of mutated subunits expressed in heterologous cells have indicated parts of the subunits involved in ATP binding, ion permeation (including calcium permeability), and membrane trafficking. We review our current understanding of the molecular properties of P2X receptors, including how this understanding is informed by the identification of distantly related P2X receptors in simple eukaryotes.

Pannexin 1 forms an anion-selective channel.

Pannexin 1 (Panx1) is expressed in various mammalian tissues including the brain and immune cells. Here, we present evidence that Panx1 when expressed in mammalian cells, forms anion-selective channels, with a rank order of permeabilities: NO (3) (-)> I(-) > Br (-)> Cl (-) > F (-)>> aspartate (-)≈ glutamate (-)≈ gluconate(-). Single-channel Panx1-mediated currents have a unitary conductance around 68 pS. Our results show that Panx1 assembles into a membrane anion channel with a relatively low single-channel conductance.

Membrane phosphatidylserine distribution as a non-apoptotic signalling mechanism in lymphocytes.

Phosphatidylserine (PS) exposure is normally associated with apoptosis and the removal of dying cells. We observed that PS is exposed constitutively at high levels on T lymphocytes that express low levels of the transmembrane tyrosine phosphatase CD45RB. CD45 was shown to be a negative regulator of PS translocation in response to various signals, including activation of the ATP receptor P2X(7). Changes in PS distribution were shown to modulate several membrane activities: Ca(2+) and Na(+) uptake through the P2X(7) cation channel itself; P2X(7)-stimulated shedding of the homing receptor CD62L; and reversal of activity of the multidrug transporter P-glycoprotein. The data identify a role for PS distribution changes in signal transduction, rapidly modulating the activities of several membrane proteins. This seems to be an all-or-none effect, coordinating the activity of most or all the molecules of a target protein in each cell. The data also suggest a new approach to circumventing multidrug resistance.

P2X(7) receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases.

The ATP-gated P2X(7) receptor (P2X(7)R) is a promising therapeutic target in chronic inflammatory diseases with highly specific antagonists currently under clinical trials for rheumatoid arthritis. Anti-inflammatory actions of P2X(7)R antagonists are considered to result from inhibition of P2X(7)R-induced release of proinflammatory cytokines from activated macrophages. However, P2X(7)Rs are also expressed in resting macrophages, suggesting that P2X(7)R may also signal via cytokine-independent mechanisms involved in joint disease. In this study, we examined P2X(7)R function in resting human lung macrophages and mouse bone marrow-derived macrophages and found that ATP induced rapid release of the lysosomal cysteine proteases cathepsin B, K, L, and S and that was independent of the presence of the proinflammatory cytokines IL-1beta and IL-18. Cathepsins released into the medium were effective to degrade collagen extracellular matrix. ATP-induced cathepsin release was abolished by P2X(7)R antagonists, absent from P2X(7)R(-/-) mouse macrophages, and not associated with cell death. Our results suggest P2X(7)R activation may play a novel and direct role in tissue damage through release of cathepsins independently of its proinflammatory actions via IL-1 cytokines.

C-terminal calmodulin-binding motif differentially controls human and rat P2X7 receptor current facilitation.

P2X(7) receptors (P2X(7)R) are ATP-gated calcium-permeable cationic channels structurally unique among the P2X family by their much longer intracellular C-terminal tail. P2X(7)Rs show several unusual biophysical properties, in particular marked facilitation of currents and leftward shift in agonist affinity in response to repeated or prolonged agonist applications. We previously found the facilitation at rat P2X(7)R resulted from a Ca(2+)-calmodulin-dependent process and a distinct calcium-independent process. However, P2X(7)Rs show striking species differences; thus, this study compared the properties of ATP-evoked facilitation of currents in HEK293 cells transiently expressing the human or rat P2X(7)R as well as rat/human, human/rat chimeric, and mutated P2X(7)Rs. Facilitation at the human P2X(7)R was 5-fold slower than at the rat P2X(7)R. Facilitation did not resulting from an increase of receptor addressing the plasma membrane. We found the human P2X(7)R shows only calcium-independent facilitation with no evidence for calmodulin-dependent processes, nor does it contain the novel 1-5-16 calmodulin binding domain present in the C terminus of rat P2X(7)R. Replacement of three critical residues of this binding domain from the rat into the human P2X(7)R (T541I, C552S, and G559V) reconstituted the Ca(2+)-calmodulin-dependent facilitation, leaving the calcium-independent facilitation unaltered. The leftward shift in the ATP concentration-response curve with repeated agonist applications appears to be a property of the calcium-independent facilitation process because it was not altered in any of the chimeric or mutated P2X(7)Rs. The absence of Ca(2+)-dependent facilitation at the human P2X(7)R may represent a protective adaptation of the innate immune response in which P2X(7)R plays significant roles.

Dynamic regulation of the P2X4 receptor in alveolar macrophages by phagocytosis and classical activation.

ATP-gated P2X(4) receptors (P2X(4)R) in macrophages and microglia have been implicated in neuropathic and inflammatory pain by currently unidentified mechanisms. P2X(4)R are found predominantly in intracellular lysosomal compartments but can be rapidly trafficked to the surface membrane by procedures that induce endolysosomal secretion. We studied total and surface membrane P2X(4)R protein expression by Western blot and biotinylation assays and functional expression by whole-cell patch clamp assays in human and rat alveolar macrophages in response to phagocytosis of zymosan and opsonized zymosan bioparticles and to classical and alternative macrophage activation. Unstimulated macrophages showed high total protein expression but very low functional expression. Phagocytosis rapidly (within 4 h) increased functional P2X(4)R expression by 2- to 7-fold as did chloroquine, an agent known to induce lysosomal secretion. In contrast, classical activation of macrophage for 48 h with IFN-gamma and TNF-alpha or IFN-gamma and LPS reduced surface and functional P2X(4)R expression by 3-fold without altering total P2X(4)R protein levels. Alternative activation with IL-4 or IL-13 did not alter total, surface or functional expression of P2X(4)R. This is the first study of the regulation of P2X(4)R in macrophages by physiological stimuli and presents a picture whereby P2X(4)R become functional in response to initial phagocytic stimuli but return to a non-functional state during sustained activation by classical macrophage activation.

Facilitation of P2X7 receptor currents and membrane blebbing via constitutive and dynamic calmodulin binding.

The ATP-gated P2X(7) receptor (P2X(7)R) is a highly unusual calcium-permeable cationic channel in that within seconds of its activation, dramatic and reversible cytoskeletal rearrangements with prominent membrane blebbing occurs. Agonist-induced membrane currents at hyperpolarized potentials show pronounced facilitation during the initial 30-100 s of receptor activation but mechanisms responsible have not been elucidated. We measured facilitation of ATP-gated currents in HEK cells expressing rat P2X(7)R and delineated distinct calcium-dependent and independent processes. The calcium-dependent facilitation was composed of an instantaneous (millisecond time domain) and slowly developing (time constant, 20 s with maximum agonist stimulation) component. Both components were prevented when recording with a highly specific calmodulin (CaM) inhibitory peptide but only the instantaneous component was reduced by expression of the dominant-negative EF-handless CaM mutant. Coimmunoprecipitation assays detected low levels of CaM binding to unstimulated P2X(7)R, and this increased by 50% during 45 s stimulation of the receptor. We identified a novel 1-5-16 Ca(2+)-dependent CaM binding motif in the intracellular C terminus of P2X(7)R; mutations in this domain resulted in the absence of calcium-dependent facilitation and binding of CaM to unstimulated or stimulated receptor. Blockade of CaM binding also delayed membrane blebbing by threefold. Our results demonstrate that CaM binds constitutively to closed P2X(7)R channels and dynamically during channel activation to significantly enhance and prolong calcium entry. This is the first example of CaM deregulating, rather than tightly controlling, calcium entry through an ion channel.

Pharmacological characterization of pannexin-1 currents expressed in mammalian cells.

Pannexin (Panx) 1 is a widely expressed protein that shares structural, but not amino acid, homology with gap junction proteins, the connexins. Panx1 does not form gap junctions in mammalian cells, but it may function as a plasma membrane hemichannel. Little is known of the pharmacological properties of panx1 expression in mammalian cells. Here, we identify three variants in the human PANX1 gene. We expressed these variants and mouse Panx1 in mammalian cells and compared Panx1-induced currents. All human Panx1 variants and the mouse Panx1 showed identical protein expression levels, localization patterns, and functional properties, although the frequency of functional expression was species-dependent. Panx1 currents were independent of changes in extracellular or intracellular calcium or phospholipase C transduction. We found compounds that inhibited Panx1 currents with a rank order of potency: carbenoxolone > disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) approximately disodium 4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate approximately 5-nitro-2-(3-phenylpropylamino)benzoic acid > indanyloxyacetic acid 94 >> probenecid >> flufenamic acid = niflumic acid. Triphosphate nucleotides (ATP, GTP, and UTP) rapidly and reversibly inhibited Panx1 currents via mechanism(s) independent of purine receptors. When Panx1 was coexpressed with purinergic P2X(7) receptor (P2X(7)R), DIDS was found to act as a P2X(7)R antagonist to inhibit ATP-evoked currents, but none of the other compounds inhibited P2X(7)R currents. This is the first detailed pharmacological characterization of Panx1-mediated currents in mammalian cells and sheds new, although contradictory, light on the hypothesis that Panx1 acts as a hemichannel to allow passage of large molecules in response to P2X(7)R activation.

Identification of key residues coordinating functional inhibition of P2X7 receptors by zinc and copper.

P2X(7) receptors are distinct from other ATP-gated P2X receptors in that they are potently inhibited by submicromolar concentrations of zinc and copper. The molecular basis for the strong functional inhibition by zinc and copper at this purinergic ionotropic receptor is controversial. We hypothesized that it involves a direct interaction of zinc and copper with residues in the ectodomain of the P2X(7) receptor. Fourteen potential metal interacting residues are conserved in the ectodomain of all mammalian P2X(7) receptors, none of which is homologous to previously identified sites in other P2X receptors shown to be important for functional potentiation by zinc. We introduced alanine substitutions into each of these residues, expressed wild-type and mutated receptors in human embryonic kidney 293 cells, and recorded resulting ATP and BzATP-evoked membrane currents. Agonist concentration-response curves were similar for all 12 functional mutant receptors. Alanine substitution at His(62) or Asp(197) strongly attenuated both zinc and copper inhibition, and the double mutant [H62A/D197A] mutant receptor was virtually insensitive to inhibition by zinc or copper. Thus, we conclude that zinc and copper inhibition is due to a direct interaction of these divalent cations with ectodomain residues of the P2X(7) receptor, primarily involving combined interaction with His(62) and Asp(197) residues.

Characterization of ATP-gated P2X7 receptors in fish provides new insights into the mechanism of release of the leaderless cytokine interleukin-1 beta.

Mammalian interleukin-1beta (IL-1beta) is produced as a biologically inactive precursor molecule, which is proteolytically cleaved to an active form by IL-1beta-converting enzyme (ICE) after the activation of P2X(7) receptor by extracellular ATP. The mechanism of IL-1beta release in non-mammalian vertebrates is largely unknown, although most of the IL-1beta gene sequences lack a conserved ICE recognition site. Here we have cloned the P2X(7) receptor from the bony fish seabream and compared agonist and antagonist profiles at this and other non-mammalian P2X(7) receptors expressed in HEK cells, as well in seabream SAF-1 cells expressing endogenous P2X(7) receptors. We used this information to further investigate the mechanisms of IL-1beta release induced by mammalian and fish P2X(7) receptors. Despite phosphatidylserine externalization and cell permeabilization in seabream leukocytes after the addition of high BzATP concentrations, IL-1beta remained unprocessed within the cell. However, activation of rat P2X(7) receptors ectopically expressed in HEK293 together with human ICE led to the specific secretion of unprocessed seabream IL-1beta. In contrast, neither seabream nor zebrafish P2X(7) receptors induced the secretion of mammalian or fish IL-1beta when expressed in HEK293, while a chimeric receptor harboring the ATP-binding domain of seabream P2X(7) and the intracellular region of its rat counterpart did so. These findings indicate that P2X(7) receptor-mediated activation of ICE and release of IL-1beta result from different downstream signaling pathways and suggest that although the mechanisms involved in IL-1beta secretion are conserved throughout evolution, distinct inflammatory signals have been selected for the secretion of this cytokine in different vertebrates.

Subunit arrangement in P2X receptors.

ATP-gated ionotropic receptors (P2X receptors) are distributed widely in the nervous system. For example, a hetero-oligomeric receptor containing both P2X2 and P2X3 subunits is involved in primary afferent sensation. Each subunit has two membrane-spanning domains. We have used disulfide bond formation between engineered cysteines to demonstrate close proximity between the outer ends of the first transmembrane domain of one subunit and the second transmembrane domain of another. After expression in HEK 293 cells of such modified P2X2 or P2X4 subunits, the disulfide bond formation is evident because an ATP-evoked channel opening requires previous reduction with dithiothreitol. In the hetero-oligomeric P2X2/3 receptor the coexpression of doubly substituted subunits with wild-type partners allows us to deduce that the hetero-oligomeric channel contains adjacent P2X3 subunits but does not contain adjacent P2X2 subunits. The results suggest a "head-to-tail" subunit arrangement in the quaternary structure of P2X receptors and show that a trimeric P2X2/3 receptor would have the composition P2X2(P2X3)2.

Reanalysis of P2X7 receptor expression in rodent brain.

P2X receptors are cationic-selective ion channels gated by extracellular ATP. There are seven subunits (P2X1-7), the first six of which are expressed throughout the peripheral and central nervous systems. P2X7 receptors are rapidly upregulated and activated as a result of inflammatory stimuli in immune cells, where they act not only as cationic channels but uniquely couple with rapid release of proinflammatory cytokines, cytoskeletal rearrangements, and apoptosis or necrotic cell death. The P2X7 receptor has been termed the cytolytic non-neuronal P2X receptor because it had not been detected in neurons until recently when it has been immunolocalized to several brain regions, particularly the hippocampus, and has been suggested to be involved in presynaptic modulation of transmitter release. Because its expression in brain neurons may have substantial functional implications, we have performed detailed immunocytochemical, immunoblot, and immunoprecipitation studies on brain and non-neuronal tissue using all currently available antibodies. We first examined rats, but staining patterns were inconsistent among antibodies; we therefore studied mice for which there are two P2X7 knock-out mice constructs available, one expressing the LacZ transgene. We found that P2X7 receptor protein is strongly and reliably detected in the submandibular gland and lung of wild-type mice but not in either of the P2X7-/- mice. However, we failed to find evidence for P2X7 receptor protein in hippocampal neurons or their input-output projections. Either the P2X7 protein in the hippocampus is below the limits of detection by the currently available methods or it is not present.

P2X4, P2Y1 and P2Y2 receptors on rat alveolar macrophages.

ATP receptors present on rat alveolar macrophages (NR8383 cells) were identified by recordings of membrane current, measurements of intracellular calcium, RT-PCR and immunocytochemistry. In whole-cell recordings with a sodium-based internal solution, ATP evoked an inward current at -60 mV. This reversed at 0 mV. The EC50 for ATP was 18 microM in normal external solution (calcium 2 mm, magnesium 1 mm). The currents evoked by 2',3-O-(4-benzoyl)benzoyl-ATP were about five-fold smaller than those observed with ATP. ADP, UTP and alphabeta-methylene-ATP (alphabetameATP) (up to 100 microM) had no effect. ATP-evoked currents were potentiated up to ten-fold by ivermectin and were unaffected by suramin (30-100 microM), pyridoxal-phosphate-6-azophenyl-(2,4-sulphonic acid) (30-100 microM), and brilliant blue G (1 microM). In whole-cell recordings with a potassium-based internal solution and low EGTA (0.01 mm), ATP evoked an inward current at -60 mV that was followed by larger outward current. ADP and UTP (1-100 microM) evoked only outward currents; these reversed polarity at the potassium equilibrium potential and were blocked by apamin (10 nm). Outward currents were also blocked by the phospholipase C inhibitor U73122 (1 microM), and they were not seen with higher intracellular EGTA (10 mm). Suramin (30 microM) blocked the outward currents evoked by ATP and UTP, but not that evoked by ADP. PPADS (10 microM) blocked the ADP-evoked outward current without altering the ATP or UTP currents. RT-PCR showed transcripts for P2X subunits 1, 4 and 7 (not 2, 3, 5, 6) and P2Y receptors 1, 2, 4 and 12 (not 6). Immunocytochemistry showed strong P2X4 receptor expression partly associated with the membrane, weak P2X7 staining that was not associated with the cell membrane, and no P2X1 receptor immunoreactivity. We conclude that rat alveolar macrophages express (probably homomeric) P2X4 receptors, but find no evidence for other functional P2X subtypes. The P2Y receptors are most likely P2Y1 and P2Y2 and these couple through phospholipase C to an increase in intracellular calcium and the opening of SK type potassium channels.

Kinetics of antagonist actions at rat P2X2/3 heteromeric receptors.

1. Currents through heteromeric P2X(2/3) receptors were evoked by applying alpha,beta-methylene-ATP to human embryonic kidney cells transfected with cDNAs encoding the P2X(2) and P2X(3) subunits. The concentration of alpha,beta-methylene-ATP were < or =30 microM because higher concentrations can activate homomeric P2X(2) receptors. The kinetics of action of three structurally unrelated antagonists were studied; these were 2', 3'-O-(2,4,6,trinitrophenyl)-ATP (TNP-ATP), pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and suramin. The association and dissociation rate constants were determined by pre-applying the antagonist for various periods prior to the co-application of agonist and antagonist, or by changing the solution from one containing only the agonist to one containing both agonist and antagonist. The high affinity of TNP-ATP for the P2X(2/3) receptor (K(D) approximately 2 nM) results from fast binding (k(+1) approximately 100 microM(-1) s(-1)) rather than slow unbinding (k(-1) approximately 0.3 s(-1)). For suramin (K(D) approximately 1 microM) the association rate constant ( approximately 1 microM(-1) s(-1)) was 100 times slower than that of TNP-ATP but the dissociation rate constant was similar (k(-1) approximately 1 s(-1)). PPADS (K(D) approximately 0.1 microM) associated and dissociated some 100 - 10,000 times more slowly than the other antagonists.

Interaction between cysteines introduced into each transmembrane domain of the rat P2X2 receptor.

1 ATP-gated ion channels (P2X receptors) contain two hydrophobic segments that are presumed to span the plasma membrane (TM1 and TM2). Pairs of cysteines were introduced by mutagenesis into the rat P2X(2) receptor, one in TM1 one in TM2, at positions where single substitutions have previously been shown to confer sensitivity to methanethiosulfonates. The receptors were expressed in HEK293 cells; interactions between the cysteines were sought by measuring the effects on ionic currents of dithiothreitol and methanethiosulfonates. 2 Nine pairs gave normally functioning channels: F44C/I328C, F44C/N333C, F44C/L338C, Q37C/I328C, Q37C/N333C, Q37C/T336C, Q37C/L338C, G30C/I328C, G30C/N333C. None formed functionally detectable disulfide bonds. 3 Currents at the F44C/L338C receptor had time course and ATP-sensitivity similar to those for the F44C mutation alone. Methyl-methanethiosulfonate bound to L338C but did not inhibit ionic current. Methyl-methanethiosulfonate inhibited currents at F44C, but not at F44C/L338C. 4 Ethylammonium-methylthiosulfonate inhibited currents at both F44C and L338C, but not at F44C/L338C. It reversed the rapid current deactivation at F44C/L338C. 5 The results suggest that a methanethiosulfonate binding to L338C prevents binding to F44C; this might indicate proximity of these two residues.

Single nucleotide polymorphisms that were identified in affective mood disorders affect ATP-activated P2X7 receptor functions.

Genetic linkage studies have previously identified many single non-synonymous nucleotide polymorphisms (SNPs) in the human P2RX7 gene in individuals with affective mood disorders. The P2RX7 gene encodes the P2X(7) receptor (P2X(7)R) that operates as an ATP-activated Ca(2+)-permeable cationic channel and induces formation of a large pore, the two functional properties that are critical for the physiological and pathological roles of the receptor. The current knowledge regarding the effects of SNPs on the P2X(7)R functional properties, which is indispensable to help elucidate the disease mechanism, is limited. In this study, we introduced by site-directed mutagenesis twelve SNP mutations in the human P2X(7) receptor that were previously identified in or associated with affective mood disorders, expressed the resultant mutants in human embryonic kidney cells, and characterized their functional properties by electrophysiology. All mutations except Q460R gave rise to profound effects on the P2X(7)R function. G150R, E186K and I568N conferred complete loss of function. V76A, R117W, L191P, T357S and E496A resulted in strong impairment of, whereas H155Y and A348T caused significant increase in, both ATP-activated ion channel function and pore formation. Q521H reduced the receptor's sensitivity to extracellular Ca(2+) inhibition. An atomic structure model of the human P2X(7)R, based on the crystal structure of the zebrafish P2X(4) receptor, suggests that the SNP mutational effects may result from changes in subunit interaction, agonist binding and/or channel gating. These results provide essential knowledge for a better understanding of the relationships between human P2RX7 SNPs and associated pathologies as well as the receptor structure-function relationships.

The P2X(7) receptor-pannexin connection to dye uptake and IL-1beta release.

The P2X(7) receptor (P2X(7)R) is uniquely associated with two distinct cellular responses: activation of a dye-permeable pathway allowing passage of molecules up to 900 Da and rapid release of the pro-inflammatory cytokine, interleukin-1beta (IL-1beta), from activated macrophage. How this dye uptake path forms and whether it is involved in IL-1beta release has not been known. Pannexin-1 is a recently identified protein found to physically associate with the P2X(7)R. Inhibition of pannexin-1 does not alter P2X(7)R ion channel activation or associated calcium flux but blocks one component of P2X(7)R-induced dye uptake and unmasks a slower, previously undetected, dye uptake pathway. Inhibition of pannexin-1 blocks P2X(7)R-mediated IL-1beta release from macrophage as well as release mediated by other stimuli which couple to activation of capase-1 and additionally inhibits the release of interleukin-1alpha, a member of the IL-1 family whose processing does not require caspase-1 activation. Thus, pannexin-1 is linked to both dye uptake and IL-1beta release but via distinct mechanisms.

Nanoparticles can cause DNA damage across a cellular barrier.

The increasing use of nanoparticles in medicine has raised concerns over their ability to gain access to privileged sites in the body. Here, we show that cobalt-chromium nanoparticles (29.5 +/- 6.3 nm in diameter) can damage human fibroblast cells across an intact cellular barrier without having to cross the barrier. The damage is mediated by a novel mechanism involving transmission of purine nucleotides (such as ATP) and intercellular signalling within the barrier through connexin gap junctions or hemichannels and pannexin channels. The outcome, which includes DNA damage without significant cell death, is different from that observed in cells subjected to direct exposure to nanoparticles. Our results suggest the importance of indirect effects when evaluating the safety of nanoparticles. The potential damage to tissues located behind cellular barriers needs to be considered when using nanoparticles for targeting diseased states.

P2X7 receptor differentially couples to distinct release pathways for IL-1beta in mouse macrophage.

The proinflammatory IL-1 cytokines IL-1alpha, IL-1beta, 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 P2X(7) receptors (P2X(7)R) by extracellular ATP is a key physiological inducer of rapid IL-1beta release 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 P2X(7)R(-/-) mice and found that release of IL-1alpha, IL-18, as well as IL-1beta, by ATP resulted exclusively from activation of P2X(7)R, release of all these IL-1 cytokines involved pannexin-1 (panx1), and that there was both a panx1-dependent and -independent component to IL-1beta 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-1beta independently of panx1 but do not release mature IL-1beta because they do not express apoptotic speck-like protein with a caspase-activating recruiting domain 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-1beta that was selectively blocked by inhibition of caspase-1 or panx1, and a calcium-independent, caspase-1/panx1-independent release of pro-IL-1beta 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-1beta.